Role of IL3RA in a Family with Lumbar Spinal Stenosis.

Lumbar spinal stenosis (LSS) is a degenerative spinal condition characterized by the narrowing of the spinal canal, resulting in low back pain (LBP) and limited leg mobility. Twin and family studies have suggested that genetics contributes to disease progression. However, the genetic causes of familial LSS remain unclear. We performed whole-exome and direct sequencing on seven female patients from a Han Chinese family with LBP, among whom four developed LSS. Based on our genetic findings, we performed gene knockdown studies in human chondrocytes to study possible pathological mechanisms underlying LSS. We found a novel nonsense mutation, c.417C > G (NM_002183, p.Y139X), in IL3RA, shared by all the LBP/LSS cases. Knockdown of IL3RA led to a reduction in the total collagen content of 81.6% in female chondrocytes and 21% in male chondrocytes. The expression of MMP-1, -3, and/or -10 significantly increased, with a more pronounced effect observed in females than in males. Furthermore, EsRb expression significantly decreased following IL3RA knockdown. Moreover, the knockdown of EsRb resulted in increased MMP-1 and -10 expression in chondrocytes from females. We speculate that IL3RA deficiency could lead to a reduction in collagen content and intervertebral disk (IVD) strength, particularly in females, thereby accelerating IVD degeneration and promoting LSS occurrence. Our results illustrate, for the first time, the association between IL3RA and estrogen receptor beta, highlighting their importance and impact on MMPs and collagen in degenerative spines in women.

Smurf1 Facilitates Oxidative Stress and Fibrosis of Ligamentum Flavum by Promoting Nrf2 Ubiquitination and Degradation.

Lumbar spinal stenosis (LSS), which can lead to irreversible neurologic damage and functional disability, is characterized by hypertrophy and fibrosis in the ligamentum flavum (LF). However, the underlying mechanism is still unclear. In the current study, the effect of Smurf1, a kind of E3 ubiquitin ligase, in promoting the fibrosis and oxidative stress of LF was investigated, and its underlying mechanism was explored. The expression of oxidative stress and fibrosis-related markers was assessed in the tissue of lumbar spinal stenosis (LSS) and lumbar disc herniation (LDH). Next, the expression of the top 10 E3 ubiquitin ligases, obtained from Gene Expression Omnibus (GEO) dataset GSE113212, was assessed in LDH and LSS, and confirmed that Smurf1 expression was markedly upregulated in the LSS group. Furthermore, Smurf1 overexpression promotes the fibrosis and oxidative stress of LF cells. Subsequently, NRF2, an important transcription factor for oxidative stress and fibrosis, was predicted to be a target of Smurf1. Mechanistically, Smurf1 directly interacts with Nrf2 and accelerates Nrf2 ubiquitination and degradation. In conclusion, the current study suggests that Smurf1 facilitated the fibrosis and oxidative stress of LF and induced the development of LSS by promoting Nrf2 ubiquitination and degradation.

EGF Contributes to Hypertrophy of Human Ligamentum Flavum via the TGF-ß1/Smad3 Signaling Pathway.

Background: The most common spinal disorder in elderly is lumbar spinal canal stenosis (LSCS). Previous studies showed that ligamentum flavum hypertrophy (LFH) with fibrosis as the main pathological change is one of the pathogenic factors leading to LSCS. Epidermal Growth Factor (EGF) is known to have an intimate relationship with fibrosis in various tissues. Nevertheless, currently, there are few studies regarding EGF in LFH. The effect of EGF on the development of LFH is unknown, and the underlying pathomechanism remains unclear. In this study, we investigated the role of EGF in LFH and its potential molecular mechanism. Methods: First, the expression levels of EGF, phosphorylation of EGF receptor (pEGFR), Transforming growth factor-ß1 (TGF-ß1), Phosphorylated Smad3 (pSmad3), collagen I and collagen III were examined via immunohistochemistry and Western blot in LF tissues from patients with LSCS or Non-LSCS. Second, primary LF cells were isolated from adults with normal LF thickness and were cultured with different concentrations of exogenous EGF with or without erlotinib/TGF-ß1-neutralizing antibody. Results: The results showed that EGF, pEGFR, TGF-ß1, pSmad3, collagen I and collagen III protein expression in the LSCS group was significantly higher than that in the Non-LSCS group. Meanwhile, pEGFR, TGF-ß1, pSmad3, collagen I and collagen III protein expression was significantly enhanced in LF cells after exogenous EGF exposure, which can be notably blocked by erlotinib. In addition, pSmad3, collagen I and collagen III protein expression was blocked by TGF-ß1-neutralizing antibody. Conclusions: EGF promotes the synthesis of collagen I and collagen III via the TGF-ß1/Smad3 signaling pathway, which eventually contributes to LFH.

Jmjd3 Mediates Neuropathic Pain by Inducing Macrophage Infiltration and Activation in Lumbar Spinal Stenosis Animal Model.

Lumbar spinal stenosis (LSS) is a major cause of chronic neuropathic back and/or leg pain. Recently, we demonstrated that a significant number of macrophages infiltrated into the cauda equina after compression injury, causing neuroinflammation, and consequently mediating neuropathic pain development and/or maintenance. However, the molecular mechanisms underlying macrophage infiltration and activation have not been elucidated. Here, we demonstrated the critical role of histone H3K27 demethylase Jmjd3 in blood-nerve barrier dysfunction following macrophage infiltration and activation in LSS rats. The LSS rat model was induced by cauda equina compression using a silicone block within the epidural spaces of the L5-L6 vertebrae with neuropathic pain developing 4 weeks after compression. We found that Jmjd3 was induced in the blood vessels and infiltrated macrophages in a rat model of neuropathic pain. The blood-nerve barrier permeability in the cauda equina was increased after compression and significantly attenuated by the Jmjd3 demethylase inhibitor, GSK-J4. GSK-J4 also inhibited the expression and activation of MMP-2 and MMP-9 and significantly alleviated the loss of tight junction proteins and macrophage infiltration. Furthermore, the activation of a macrophage cell line, RAW 264.7, by LPS was significantly alleviated by GSK-J4. Finally, GSK-J4 and a potential Jmjd3 inhibitor, gallic acid, significantly inhibited mechanical allodynia in LSS rats. Thus, our findings suggest that Jmjd3 mediates neuropathic pain development and maintenance by inducing macrophage infiltration and activation after cauda equina compression and thus may serve as a potential therapeutic target for LSS-induced neuropathic pain.

The correlation between imaging expression of P16 and S100 in hypertrophic ligamentum flavum.

BACKGROUND: Lumbar spinal stenosis (LSS) is a common degenerative disease, which can lead to neurological dysfunction and requires surgical treatment. In the previous study, we used H&E staining and immunohistochemistry to qualitatively analyze the expression of S100 and P16 in the pathological process of ligamentum flavum (LF) hypertrophy in patients with LSS. To further explore the relationship between P16, S100 and LF hypertrophy in patients with LSS, we quantitatively detected S100 and P16 and their expressed products based on molecular biology techniques, and analyzed their imaging correlation. METHODS: Before posterior lumbar surgery, LF thickness was measured by Magnetic Resonance Imaging (MRI). Through the operation, we obtained the specimens of LF from 120 patients, all of whom were L4/5 LF. They were designated: simple lumbar disc herniation (LDH), single-segment spinal stenosis (SLSS), and double-segment LSS (DLSS). The detection of each side of LF was assessed. S100 and P16 and their expression products were detected by western blot and quantitative polymerase chain reaction (qPCR). RESULTS: The dorsal mRNA expression of P16 in DLSS group was significantly higher than that in SLSS group. On the dorsal and dural side of LF, the expression of P16 mRNA and proteins in the LDH group was significantly lower than that in SLSS and DLSS groups. We found a correlation between the thickness of LF and the expression of P16. However, there was no significant difference in the expression of S100 mRNA and S100 protein on both sides of the ligament and among the three groups, and no significant correlation between the expression of S100 and the thickness of LF. CONCLUSIONS: P16 is involved in the process of LF hypertrophy in patients with LSS, and the imaging thickness of LF is related to the expression of P16. No obvious evidence proves that S100 may be related to the hypertrophy of LF in patients with LSS.

Effects of SHINBARO2 on Rat Models of Lumbar Spinal Stenosis.

Lumbar spinal stenosis (LSS) is a major cause of chronic low back pain; however, only a few therapies which have been used in clinics still have limited effects on functional recovery. SHINBARO2 is a refined traditional formulation for inflamed lesions and relieve pain of muscular skeletal disease. This study aimed at investigating the effects of SHINBARO2 on LSS and at determining its underlying molecular mechanism in rat models. The LSS rat models were set up by surgical operations in 6-week-old male Sprague-Dawley rats. SHINBARO2 was orally or intraperitoneally administered for 14 days. The motor and sensory ability of rats were evaluated using the activity cage and hot plate method. On the termination day, total vertebrae including the disc and spinal cord were excised for ex vivo study. SHINBARO2 improved locomotor functions and pain sensitivity in LSS rat models. Mechanism study suggested that SHINBARO2 inhibited the production of nitric oxide and prostaglandin E2 in tissues from LSS-induced rats. SHINBARO2 also suppressed the expression of proinflammatory cytokines including tumor necrosis factor-α and interleukin-1ß. The activation of NF-κB by LSS surgery was effectively reduced by SHINBARO2, which coincided with the inhibition of IκB degradation. In addition, brain-derived neurotrophic factor (BDNF), a potent promoter of neurite growth, and its downstream ERK signaling were also regulated by SHINBARO2. These findings suggest that the effect of SHINBARO2 might be associated in part with the anti-inflammation and pain control in LSS rat models.

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.

The expression of P16 and S100 associated with elastin degradation and fibrosis of the Ligamentum Flavum hypertrophy.

BACKGROUND: One of the characteristics of lumbar spinal stenosis (LSS) is elastin degradation and fibrosis in the ligamentum flavum (LF). However, the biochemical factors that cause these histologic changes is unclear. P16 and S100 participate in scar formation and collagen development in wound healing and fibrosis diseases. In this study, we investigate the association between P16 and S100 expression and the fibrosis of the hypertrophic LF in LSS. METHODS: The LF specimens were surgically obtained from 30 patients with single-segment LSS (SLSS), 30 patients with double-segment LSS (DLSS) and 30 patients with L4/5 lumbar disc herniation (LDH). The LF thickness was measured by axial T1-weighted MRI. The extent of LF elastin degradation and fibrosis were graded based on hematoxylin-eosin (HE) and Verhoff's Van Gieson's (VVG) stain, respectively. The localization of P16 and S100 was determined by immunohistochemistry. RESULTS: The Absolute and relative LF thickness were greater in the DLSS group compared with the SLSS and LDH groups (p <  0.05). The elastic tissue from the dorsal aspect to the dural aspect in SLSS and DLSS groups was significantly increased. The amount of collagen deposition and elastic tissue is significantly higher in the DLSS group compared with the SLSS and LDH groups (p <  0.05). The specimens in the DLSS group showed positive staining of P16, especially in the dorsal layer. Almost all samples in the SLSS group were partially positive for P16. The LDH group showed negative staining of P16 in both the dural and dorsal layers. All the three groups were stained with S100 in the dorsal layer of the LF. On the contrary, S100 staining was absent in the dural layer of the LF in the three groups. CONCLUSIONS: Elastin degradation and fibrosis of the LF in the DLSS patients is more severe compared with the SLSS and LDH patients. Increased expression of P16 associated with LF fibrosis and thickness, suggested that the expression of P16 may related to LF hypertrophy in the patients who suffer with LSS. LF hypertrophy process may not be associated with high expression of S100.

Defective autophagy in vascular smooth muscle cells enhances atherosclerotic plaque instability.

Autophagy is considered as an evolutionarily conserved cellular catabolic process. Defective autophagy has been implicated in various human diseases, including cardiovascular diseases. Recently, we and others demonstrated that defective autophagy in vascular smooth muscle cells (SMCs) promotes the progression of atherosclerosis. In this study, we investigated the role of autophagy in SMCs on plaque instability in vivo. We generated mice with a defect atg7in which is an essential gene for autophagy, in SMCs by crossing Atg7f/f mice with transgelin (Tagln) Cre+/0 mice (Atg7cKO). Then, Atg7cKO and apolipoprotein E (apoe)-deficient (apoeKO) mice were crossed to generate Atg7cKO:apoeKO mice. To generate a mouse model of plaque instability, we conducted to form a tandem stenosis in the carotid artery of Atg7cKO:apoeKO mice and their controls (apoeKO mice) at the age of 10 weeks. At 5 weeks after surgery, the percentage of cross-sectional stenosis area in the operated common carotid artery of Atg7cKO:apoeKO mice was significantly higher than that in apoeKO mice. In addition, thrombus, which was not observed in apoeKO mice, was frequently found in Atg7cKO:apoeKO mice. Furthermore, the number of Berlin blue staining-positive areas, which indicated intraplaque hemorrhage, was significantly higher in Atg7cKO:apoeKO mice than in control apoeKO mice. Taken together, our data suggest that defective autophagy in SMCs enhances plaque instability and the risk of plaque rupture.

Macrophage Infiltration Is a Causative Factor for Ligamentum Flavum Hypertrophy through the Activation of Collagen Production in Fibroblasts.

Ligamentum flavum (LF) hypertrophy causes lumbar spinal canal stenosis, leading to leg pain and disability in activities of daily living in elderly individuals. Although previous studies have been performed on LF hypertrophy, its pathomechanisms have not been fully elucidated. In this study, we demonstrated that infiltrating macrophages were a causative factor for LF hypertrophy. Induction of macrophages into the mouse LF by applying a microinjury resulted in LF hypertrophy along with collagen accumulation and fibroblasts proliferation at the injured site, which were very similar to the characteristics observed in the severely hypertrophied LF of human. However, we found that macrophage depletion by injecting clodronate-containing liposomes counteracted LF hypertrophy even with microinjury. For identification of fibroblasts in the LF, we used collagen type I α2 linked to green fluorescent protein transgenic mice and selectively isolated green fluorescent protein-positive fibroblasts from the microinjured LF using laser microdissection. A quantitative RT-PCR on laser microdissection samples revealed that the gene expression of collagen markedly increased in the fibroblasts at the injured site with infiltrating macrophages compared with the uninjured location. These results suggested that macrophage infiltration was crucial for LF hypertrophy by stimulating collagen production in fibroblasts, providing better understanding of the pathophysiology of LF hypertrophy.

Myofibroblast in the ligamentum flavum hypertrophic activity.

PURPOSE: Majority of the previous studies compared lumbar spinal stenosis (LSS) and lumbar disc herniation (LDH) patients for analyses of LFH. However, the separation of normal/hypertrophied LF has often been ambiguous and the severity of hypertrophic activity differed. Here, we present a novel analysis scheme for LFH in which myofibroblast is proposed as a major etiological factor for LFH study. METHODS: Seventy-one LF patient tissue samples were used for this study. Initially, mRNA levels of the samples were assessed by qRT-PCR: angiopoietin-like protein-2 (ANGPTL2), transforming growth factor-beta1 (TGF-ß1), vascular endothelial growth factor (VEGF), interleukin-6, collagen-1, 3, 4, 5, and 11, and elastin. Myofibroblasts were detected by immune stain using α-smooth muscle actin (αSMA) as a marker. To study the myofibroblast in TGF-ß pathway, LF tissues were analyzed for protein levels of αSMA/TGF-ß1 by Western blot. In addition, from LF cells cultured with exogenous TGF-ß1 conditioned medium, expression of αSMA/collagen-1 was assessed and the cell morphology was identified. RESULTS: The comparative analysis of mRNA expression levels (LSS vs LDH) failed to show significant differences in TGF-ß1 (p = 0.08); however, we found a significant positive correlation among ANGPTL2, VEGF, TGF-ß1, and collagen-1 and 3, which represent common trends in hypertrophic activity (p < 0.05). We detected myofibroblast in the patient samples by αSMA staining, and the protein levels of αSMA were positively correlated with TGF-ß1. In LF cell culture, exogenous TGF-ß1 upregulated αSMA/collagen-1 mRNA levels and facilitated trans-differentiation to myofibroblast. CONCLUSIONS: We conclude that the transition of fibroblast to myofibroblasts via TGF-ß pathway is a key linker between inflammation and fibrosis in LFH mechanism.

Amyloid deposits derived from transthyretin in the ligamentum flavum as related to lumbar spinal canal stenosis.

Amyloidosis is a protein conformational disorder with the distinctive feature of extracellular accumulation of amyloid fibrils that come from different proteins. In the ligamentum flavum of the lumbar spine, amyloid deposits were frequently found in elderly patients with lumbar spinal canal stenosis and were at least partially formed by wild-type transthyretin. However, how amyloid deposits in the ligamentum flavum affect lumbar spinal canal stenosis has remained unclear. In this study, we analyzed clinical, pathologic, and radiologic findings of patients with lumbar spinal canal stenosis who had amyloid deposits in the ligamentum flavum. We studied 95 ligamentum flavum specimens obtained from 56 patients with lumbar spinal canal stenosis and 21 ligamentum flavum specimens obtained from 19 patients with lumbar disk herniation. We evaluated histopathologic findings and clinicoradiologic manifestations, such as thickness of the ligamentum flavum and lumbar spinal segmental instability. We found that all 95 ligamentum flavum specimens resected from patients with lumbar spinal canal stenosis had amyloid deposits, which we classified into two types, transthyretin-positive and transthyretin-negative, and that transthyretin amyloid formation in the ligamentum flavum of patients with lumbar spinal canal stenosis was an age-associated phenomenon. The amount of amyloid in the ligamentum flavum was related to clinical manifestations of lumbar spinal canal stenosis, such as thickness of the ligamentum flavum and lumbar spinal segmental instability, in the patients with lumbar spinal canal stenosis with transthyretin-positive amyloid deposits. To our knowledge, this report is the first to show clinicopathologic correlations in transthyretin amyloid deposits of the ligamentum flavum. In conclusion, transthyretin amyloid deposits in the ligamentum flavum may be related to the pathogenesis of lumbar spinal canal stenosis in elderly patients.

Biomarkers related to hypertrophy of the ligamentum flavum: a systematic review of the literature.

OBJECTIVE: Spinal stenosis is one of the most common spinal disorders in the elderly. Hypertrophy of the ligamentum flavum (HLF) can contribute to spinal stenosis. The current literature suggests that various biomarkers may play important roles in the pathogenesis of HLF. However, the connection between these biomarkers and the development of HLF is still not well understood. This systematic review aims to explore the current literature on biomarkers related to the development of HLF. METHODS: A literature search was conducted using PubMed, Embase, Web of Science, and Cochrane Library. The search strategy looked for the titles, abstracts, and keywords of studies that contained a combination of the following phrases: "ligamentum flavum OR yellow ligament," "biomarkers," and "hypertrophy." Recorded data included study design, demographic characteristics (number of patients of each gender and mean age), study period, country where the study was conducted, biomarkers, and diagnostic modalities used. Risk of bias was assessed using the Newcastle-Ottawa Scale for case-control studies. RESULTS: The authors identified 39 studies. After screening, 26 full-text original articles assessing one or more biomarkers related to HLF were included. The included studies were conducted over a 22-year period. The most popular biomarkers studied, in order of frequency reported, were collagen types I and III (n = 10), transforming growth factor ß (TGF-ß) (n = 8), and interleukin (IL)-6 (n = 6). The authors found that mechanical stretching forces, tissue inhibitor of metalloproteinases 2 (TIMP-2) induction, and TGF-ß were associated with increased amounts of collagen I and III. IL-6 expression was increased by microRNA-21, as well as by leptin, through the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway. CONCLUSIONS: Biomarkers such as TGF-ß, IL-6, and collagen I and III have been consistently correlated with the development of HLF. However, the pathogenesis of HLF remains unclear due to the heterogeneity of the studies, patient populations, and research at the molecular level. Further studies are necessary to better characterize the pathogenesis of HLF and provide a more comprehensive understanding of how these biomarkers may aid in the diagnosis and treatment of HLF.

CTSD upregulation as a key driver of spinal ligament abnormalities in spinal stenosis.

Spinal stenosis (SS) is frequently caused by spinal ligament abnormalities, such as ossification and hypertrophy, which narrow the spinal canal and compress the spinal cord or nerve roots, leading to myelopathy or sciatic symptoms; however, the underlying pathological mechanism is poorly understood, hampering the development of effective nonsurgical treatments. Our study aims to investigate the role of co-expression hub genes in patients with spinal ligament ossification and hypertrophy. To achieve this, we conducted an integrated analysis by combining RNA-seq data of ossification of the posterior longitudinal ligament (OPLL) and microarray profiles of hypertrophy of the ligamentum flavum (HLF), consistently pinpointing CTSD as an upregulated hub gene in both OPLL and HLF. Subsequent RT-qPCR and IHC assessments confirmed the heightened expression of CTSD in human OPLL, ossification of the ligamentum flavum (OLF), and HLF samples. We observed an increase in CTSD expression in human PLL and LF primary cells during osteogenic differentiation, as indicated by western blotting (WB). To assess CTSD's impact on osteogenic differentiation, we manipulated its expression levels in human PLL and LF primary cells using siRNAs and lentivirus, as demonstrated by WB, ALP staining, and ARS. Our findings showed that suppressing CTSD hindered the osteogenic differentiation potential of PLL and LF cells, while overexpressing CTSD activated osteogenic differentiation. These findings identify CTSD as a potential therapeutic target for treating spinal stenosis associated with spinal ligament abnormalities.

Evaluation of differences in expression pattern of three isoforms of the transforming growth factor beta in patients with lumbosacral stenosis.

The study investigates molecular changes in the lumbosacral (L/S) spine's yellow ligamentum flavum during degenerative stenosis, focusing on the role of transforming growth factor beta 1-3 (TGF-ß-1-3). Sixty patients with degenerative stenosis and sixty control participants underwent molecular analysis using real-time quantitative reverse transcription reaction technique (RTqPCR), enzyme-linked immunosorbent assay (ELISA), Western blot, and immunohistochemical analysis (IHC). At the mRNA level, study samples showed reduced expression of TGF-ß-1 and TGF-ß-3, while TGF-ß-2 increased by only 4%. Conversely, at the protein level, the study group exhibited significantly higher concentrations of TGF-ß-1, TGF-ß-2, and TGF-ß-3 compared to controls. On the other hand, at the protein level, a statistically significant higher concentration of TGF-ß-1 was observed (2139.33 pg/mL ± 2593.72 pg/mL vs. 252.45 pg/mL ± 83.89 pg/mL; p < 0.0001), TGF-ß-2 (3104.34 pg/mL ± 1192.74 pg/mL vs. 258.86 pg/mL ± 82.98 pg/mL; p < 0.0001), TGF-ß-3 (512.75 pg/mL ± 107.36 pg/mL vs. 55.06 pg/mL ± 9.83 pg/mL, p < 0.0001) in yellow ligaments obtained from patients of the study group compared to control samples. The study did not establish a significant correlation between TGF-ß-1-3 concentrations and pain severity. The findings suggest that molecular therapy aimed at restoring the normal expression pattern of TGF-ß-1-3 could be a promising strategy for treating degenerative stenosis of the L/S spine. The study underscores the potential therapeutic significance of addressing molecular changes at the TGF-ß isoforms level for better understanding and managing degenerative spinal conditions.

MMP-1 overexpression induced by IL-1ß: possible mechanism for inflammation in degenerative lumbar facet joint.

BACKGROUND: More and more attention has been focused on the inflammation or degeneration caused by biochemical factors in radiculopathy during lumbar facet joint degeneration. This study was designed to examine the expression and relationship of MMP-1/TIMP-1 and interleukin-1ß (IL-1ß), and to analyze the possible mechanism in degenerative lumbar facet joint disease. METHODS: Lumbar facet joint cartilage and synovial tissues in 36 cases of posterior lumbar surgery were harvested to investigate IL-1ß and MMP-1/TIMP-1 by immunohistochemistry and Western blot analysis. Double labeling immunofluorescence and real-time PCR, respectively, were used to assess the relationship between IL-1ß and MMP-1. RESULTS: IL-1ß and MMP-1 were low in the lumbar disc herniation (LDH) group, and increased markedly in the lumbar spinal canal stenosis (LSCS) group (P < 0.05). However, there is no significant difference of TIMP-1 between LDH group and LSCS group (P > 0.05). Double staining results indicated that IL-1ß overlapped with MMP-1 in the LSCS group. Moreover, real-time PCR results showed that MMP-1 mRNA in chondrocytes in vitro was affected in a dose- and time-dependent manner in response to IL-1ß stimulation. CONCLUSIONS: Overexpression of MMP-1, induced by IL-1ß, plays an important role in the inflammatory process of lumbar facet joint degeneration.

Hypertrophy of the ligamentum flavum in lumbar spinal canal stenosis is associated with increased bFGF expression.

PURPOSE: A prospective study was undertaken to investigate basic fibroblast growth factor (bFGF) expression in hypertrophic ligamentum flavum (LF) from patients with lumbar spinal canal stenosis (LSCS) and to determine whether there was a correlation of bFGF expression with LF thickness. METHODS: Twenty patients with lumbar spinal canal stenosis were enrolled in this study. bFGF mRNA and protein expressions in LF were analyzed using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA), respectively. The thickness of LF was measured by axial T1-weighted magnetic resonance imaging. RESULTS: Expression of bFGF was substantially higher in the hypertrophic LF group than in the control group (P < 0.001) as quantified by quantitative real-time PCR. In immunohistochemical study, bFGF was positively stained on the fibroblasts within hypertrophic LF compared to nonpathologic LF of controls. Subsequent ELISA analysis revealed that bFGF concentration in the hypertrophic LF group was remarkably higher than that in the control group (P = 0.003). The thickness of LF in the hypertrophic LF was significantly greater than that in the control group (P < 0.001). LSCS patients with greater severity of LF hypertrophy had significantly higher bFGF levels in the LF tissues (P < 0.001). Furthermore, the bFGF concentration exhibited a positive correlation with the LF thickness (r = 0.974, P < 0.001). CONCLUSIONS: These findings suggest that the increased expression of bFGF is associated with the hypertrophy of ligamentum flavum in patients with LSCS.

Population-Based Evaluation of Total Protein in Cerebrospinal Fluid.

OBJECTIVES: To present a normal range of cerebrospinal fluid (CSF) protein levels in a community-based population and to evaluate factors that contribute to CSF protein level variability. PATIENTS AND METHODS: Samples of CSF protein were obtained from participants aged 32 to 95 years who underwent lumbar puncture (LP) between November 1, 2007, and October 1, 2017, as part of the Mayo Clinic Study of Aging, a longitudinal, population-based study of residents of Olmsted County, Minnesota. RESULTS: A total of 633 participants (58.1% male; 99.1% White; mean ± SD age, 70.9±11.6 years) underwent LP with recorded CSF protein level. Mean ± SD CSF protein level was 52.2±18.4 mg/dL (to convert to mg/L, multiply by 10), with a 95% reference interval of 24.0 to 93.4 mg/dL (range, 14.0-148.0 mg/dL). Spinal stenosis and arterial hypertension were associated with higher CSF protein levels on univariable analysis (P<.001). Increasing age, male sex, and diabetes were all independently associated with higher CSF protein levels on multivariable analysis (P<.001). In the 66 participants with repeated LPs within 2.5 years, the coefficient of repeatability was 26.1 mg/dL. Eleven participants (16.7%) had a CSF protein level difference of 20 mg/dL or more between serial LPs, and 4 (6.1%) had a difference of 25 mg/dL or more. There was a trend toward greater CSF protein level variability in patients with spinal stenosis (P=.054). CONCLUSION: This large population-based study showed that CSF protein level can vary significantly among individuals. Elevated CSF protein level was independently associated with older age, male sex, and diabetes and is higher than listed in many laboratories. These findings emphasize the necessity of evidence-based reevaluation and standardization of CSF protein metrics.

ACSM5 inhibits ligamentum flavum hypertrophy by regulating lipid accumulation mediated by FABP4/PPAR signaling pathway.

BACKGROUND: Ligamentum flavum (LF) hypertrophy is the main cause of lumbar spinal canal stenosis (LSCS). Previous studies have shown that LF hypertrophy tissue exhibits abnormal lipid accumulation, but the regulatory mechanism remains unclear. The objective of this study was to explore the function and potential mechanism of ACSM5 in LF lipid accumulation. METHODS: To assess the ACSM5 expression levels, lipid accumulation and triglyceride (TG) level in LF hypertrophy and normal tissue, we utilized RT-qPCR, western blot, oil red O staining, and TG assay kit. The pearson correlation coefficient assay was used to analyze the correlation between ACSM5 levels and lipid accumulation or TG levels in LF hypertrophy tissue. The role of ACSM5 in free fatty acids (FFA)-induced lipid accumulation in LF cells was assessed in vitro, and the role of ACSM5 in LF hypertrophy in mice was verified in vivo. To investigate the underlying mechanisms of ACSM5 regulating lipid accumulation in LF, we conducted the mRNA sequencing, bioinformatics analysis, and rescue experiments. RESULTS: In this study, we found that ACSM5, which was significantly down-regulated in LF tissues, correlated with lipid accumulation. In vitro cell experiments demonstrated that overexpression of ACSM5 significantly inhibited FFA-induced lipid accumulation and fibrosis in LF cells. In vivo animal experiments further confirmed that overexpression of ACSM5 inhibited LF thickening, lipid accumulation, and fibrosis. Mechanistically, ACSM5 inhibited lipid accumulation of LF cells by inhibiting FABP4-mediated PPARγ signaling pathway, thereby improving hypertrophy and fibrosis of LF. CONCLUSIONS: our findings elucidated the important role of ACSM5 in the regulation of LF lipid accumulation and provide insight into potential therapeutic interventions for the treatment of LF hypertrophy. This study further suggested that therapeutic strategies targeting lipid deposition may be an effective potential approach to treat LF hypertrophy-induced LSCS.

Cellularity and cartilage matrix increased in hypertrophied ligamentum flavum: histopathological analysis focusing on the mechanical stress and bone morphogenetic protein signaling.

STUDY DESIGN: Histopathological and immunohistochemical analysis. OBJECTIVE: To investigate the histological changes and expression of bone morphogenetic protein (BMP) signaling component in hypertrophied ligamentum flavum (LF), and to clarify the effect of mechanical stress on them. SUMMARY OF BACKGROUND DATA: Hypertrophic changes of the LF are a major factor in degenerative lumbar canal stenosis (DLCS), but their mechanism remains unclear. BMPs are growth factors that regulate many cellular processes including proliferation, differentiation, and extracellular matrix synthesis. However, a few studies have investigated the expressions of BMP signaling in the hypertrophied LF. METHODS: A total of 133 LF specimens from patients with DLCS and 17 control LF specimens from patients with lumbar disc herniation were analyzed histologically using hematoxylin and eosin, elastica van Gieson, and toluidine blue staining. To analyze the influence of mechanical stress, the DLCS specimens were divided into 2 groups: DLCS with and DLCS without hypermobility groups. The LF thickness was measured by magnetic resonance image, and the correlations between the thickness and the histological data were analyzed. Immunohistochemical analyses were carried out to confirm the expressions and localizations of BMP signaling components. RESULTS: The cell number and cartilage matrix area were significantly increased in the hypertrophied LF, and those changes were more obvious in DLCS with hypermobility than in DLCS without hypermobility. The cellularity and percentage of cartilage matrix area had positive linear correlations with the LF thickness. BMP receptors and BMP ligands were both expressed by many cells of the hypertrophied LF, and some of these cells were positive for Sox9, CD105, and Msx2. The percentage of immunopositive cells for each BMP receptor type was significantly higher in DLCS with hypermobility than in DLCS without hypermobility. CONCLUSIONS: Higher cellularity and increased cartilage matrix area are important changes in LF hypertrophy. These results suggest that BMP signaling and mechanical stress may play a role in the hypertrophied LF.