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.

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.

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.

Melittin regulates iron homeostasis and mediates macrophage polarization in rats with lumbar spinal stenosis.

Lumbar spinal stenosis (LSS) is defined as spinal canal narrowing, resulting in the compression of the nerves traversing the lower back into the leg. Inflammation is the most common cause of LSS. Elevated iron stores are often associated with chronic inflammation resulting in nerve damage-induced pain. Macrophage polarization to either the M1 (inflammatory) or M2 (anti-inflammatory) type is essential for regulating host defenses and promoting tissue repair. However, the precise role of macrophage polarization in iron release or retention in LSS pathophysiology remains elusive. Melittin, a component of bee venom, modulates iron metabolism-related macrophage polarization and is beneficial in LSS. We treated primary peritoneal macrophages with melittin and assessed macrophage polarization by immunofluorescence staining. Melittin (100 and 250 µg/kg) effects on iron deposition-induced macrophage polarization were also evaluated using immunochemistry, real-time PCR, and flow cytometry in an LSS rat model. Locomotor function was assessed using the Basso-Beattie-Bresnahan (BBB) locomotor rating scale, ladder scoring, and von Frey test for up to 3 weeks. Melittin induced M2 polarization of iron-insulted primary macrophages in vitro and increased the proportion of M2 macrophages in the damaged spinal cord in vivo. Moreover, melittin attenuated iron overload-induced M1 polarization by regulating iron metabolism-related genes in rats with LSS. In conclusion, melittin improves locomotor recovery and stimulates axonal growth following LSS. Additionally, it promotes functional recovery in LSS rat models by regulating macrophage iron metabolism, thereby activating M2 macrophages, suggesting its potential application in LSS treatment.

Relationship between Severity of Lumbar Spinal Stenosis and Ligamentum Flavum Hypertrophy and Serum Inflammatory Factors.

Objective: This study is aimed at investigating the correlation between lumbar spinal stenosis (LSS) severity, ligamentum flavum hypertrophy, and the upregulation of inflammatory markers. Methods: From March 2019 and May 2022, eighty-five inpatients with LSS were enlisted as the study's research group, while sixty-five patients hospitalized for lumbar intervertebral disc herniation over the same time period served as the study's control group. Moreover, mild, moderate, and severe subgroups of patients were created within the research population based on their LSS severity. The ligamentum flavum thickness and the positive expression rates of TNF-α, TGF-ß1, and IL-1α were compared between the study group and the control group. The levels of TNF-α, TGF-ß1, and IL-1α that were found to be positively expressed were compared between the mild, moderate, and severe groups. Patients with LSS had their ligamentum flavum thickness and their positive expression rates of TNF-α, TGF-ß1, and IL-1α analyzed using Spearman correlation analysis. We evaluated the diagnostic utility of the positive expression rates of IL-α1, TGF-ß1, and TNF-α and ligamentum flavum thickness in distinguishing the severity of LSS using a receiver operating characteristic (ROC) curve. Results: The rates of both lower limb pain (40.00%) and intermittent claudication (80.00%) in the LSS group were higher than those in the lumbar disc herniation group (15.38%, 12.31%), with statistical significance (P < 0.05). However, no substantial disparity was observed in left lower limb pain, right lower limb pain, low back pain, lower limb sensation, muscle strength, and reflex abnormalities between the two groups (P > 0.05). Positive expressions of TGF-ß1, TNF-α, and IL-1α and thicker ligamentum flavum were more prevalent in the LSS group than in the lumbar intervertebral disc herniation group. All indexes were significantly (P < 0.05) higher in the moderate stenosis group than in the severe stenosis group. Additionally, the thickness of the ligamentum flavum and the positive expression rates of TNF-α, TGF-ß1, and IL-1α were higher in the mild and moderate stenosis groups than in the severe stenosis group. The expression levels of TNF-α, TGF-ß1, and IL-1α were favorably linked with ligamentum flavum thickness (P < 0.05). ROC curve analysis showed that the thickness of ligamentum flavum, the expression of IL-1α, the expression of TGF-ß1, and the expression of TNF-α could effectively diagnose mild, moderate, and severe LSS (P < 0.05). Conclusion: Ligamentum flavum hypertrophy and positive expression rates of IL-1α, TGF-ß1, and TNF-α are closely linked to LSS, which can effectively identify mild, moderate, and severe LSS.

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.

Macrophage migration inhibitory factor takes part in the lumbar ligamentum flavum hypertrophy.

The present study aimed to observe the content difference of macrophage migration inhibitory factor [MIF; novoprotein recombinant human MIF (n­6his) (ch33)], TGFß1 and MMP13 in patients with and without ligamentum flavum (LF) hypertrophy and investigate the roles of MIF in LF hypertrophy. The concentration of MIF, TGFß1 and MMP13 in LF were detected by ELISA in a lumbar spinal stenosis (LSS) group and a lumbar disc herniation (LDH) group. Culture of primary LFs and identification were performed for the subsequent study. Cell treatments and cell proliferation assay by CCK­8 was performed. Western blot and quantitative PCR analysis were used to detect the expression of TGFß1, MMP13, type I collagen (COL­1) and type III collagen (COL­3) and Src which were promoted by MIF. The concentration of MIF, TGFß1 and MMP13 were higher in the LSS group compared with the LDH group. Culture of primary LFs and identification were performed. Significant difference in LFs proliferation occurred with treatment by MIF at a concentration of 40 nM for 48 h (P<0.05). The gene and protein expression of TGFß1, MMP13, COL­1, COL­3 and Src were promoted by MIF (P<0.05). Proliferation of LFs was induced by MIF and MIF­induced proliferation of LFs was inhibited by PP1 (a Src inhibitor). MIF may promote the proliferation of LFs through the Src kinase signaling pathway and can promote extracellular matrix changes by its pro­inflammatory effect. MIF and its mediated inflammatory reaction are driving factors of LF hypertrophy.

Expression of Estrogen Receptor Alpha and Evaluation of Histological Degeneration Scores in Fibroblasts of Hypertrophied Ligamentum Flavum: A Qualitative Study.

The most common spinal disorder in elderly is lumbar spinal stenosis (LSS), resulting partly from ligamentum flavum (LF) hypertrophy. Its pathophysiology is not completely understood. The present study wants to elucidate the role of estrogen receptor α (ER α) in fibroblasts of hypertrophied LF. LF samples of 38 patients with LSS were obtained during spinal decompression. Twelve LF samples from patients with disk herniation served as controls. Hematoxylin & Eosin (H&E) and Elastica stains and immunohistochemistry for ER α were performed. The proportions of fibrosis, loss and/or degeneration of elastic fibers and proliferation of collagen fibers were assessed according to the scores of Sairyo and Okuda. Group differences in the ER α and Sairyo and Okuda scores between patients and controls, male and female sex and absence and presence of additional orthopedic diagnoses were assessed with the Mann-Whitney U test. There was a tendency towards higher expression of ER α in LF fibroblasts in the hypertrophy group (p = 0.065). The Sairyo and Okuda scores were more severe for the hypertrophy group but, in general, not statistically relevant. There was no statistically relevant correlation between the expression of ER α and sex (p = 0.326). ER α expression was higher in patients with osteochondrosis but not statistically significant (p = 0.113). In patients with scoliosis, ER α expression was significantly lower (p = 0.044). LF hypertrophy may be accompanied by a higher expression of ER α in fibroblasts. No difference in ER α expression was observed regarding sex. Further studies are needed to clarify the biological and clinical significance of these findings.

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.

Integrative analysis of genome-wide DNA methylation and single-nucleotide polymorphism identified ACSM5 as a suppressor of lumbar ligamentum flavum hypertrophy.

BACKGROUND: Hypertrophy of ligamentum flavum (HLF) is a common lumbar degeneration disease (LDD) with typical symptoms of low back pain and limb numbness owing to an abnormal pressure on spinal nerves. Previous studies revealed HLF might be caused by fibrosis, inflammatory, and other bio-pathways. However, a global analysis of HLF is needed severely. METHODS: A genome-wide DNA methylation and single-nucleotide polymorphism analysis were performed from five LDD patients with HLF and five LDD patients without HLF. Comprehensive integrated analysis was performed using bioinformatics analysis and the validated experiments including Sanger sequencing, methylation-specific PCR, qPCR and ROC analysis. Furthermore, the function of novel genes in ligamentum flavum cells (LFCs) was detected to explore the molecular mechanism in HLF through knock down experiment, overexpression experiment, CCK8 assay, apoptosis assay, and so on. RESULTS: We identified 69 SNP genes and 735 661 differentially methylated sites that were enriched in extracellular matrix, inflammatory, and cell proliferation. A comprehensive analysis demonstrated key genes in regulating the development of HLF including ACSM5. Furthermore, the hypermethylation of ACSM5 that was mediated by DNMT1 led to downregulation of ACSM5 expression, promoted the proliferation and fibrosis, and inhibited the apoptosis of LFCs. CONCLUSION: This study revealed that DNMT1/ACSM5 signaling could enhance HLF properties in vitro as a potential therapeutic strategy for HLF.

Introduction and reproducibility of an updated practical grading system for lumbar foraminal stenosis based on high-resolution MR imaging.

In this paper we sought to develop and assess the reproducibility of an updated 6-point grading system for lumbar foraminal stenosis based on the widely used Lee classification that more accurately describes lumbar foraminal stenosis as seen on high-resolution MRI. Grade A indicates absence of foraminal stenosis. Grades B, C, D and E indicate presence of foraminal stenosis with contact of the nerve root with surrounding anatomical structures (on one, two, three or four sides for B, C, D and E respectively) yet without morphological change of the nerve root. To each grade, a number code indicating the location of contact between the nerve root and surrounding anatomical structure(s) is appended. 1, 2, 3 and 4 indicate contact of the nerve root at superior, posterior, inferior and anterior position of the borders of the lumbar foramen. Grade F indicates presence of foraminal stenosis with morphological change of the nerve root. Three readers graded the lumbar foramina of 101 consecutive patients using high-resolution T2w (and T1w) MR images with a spatial resolution of beyond 0.5 mm3. Interreader agreement was excellent (Cohen's Kappa = 0.866-1). Importantly, 30.6%/31.6%/32.2% (reader 1/reader 2/ reader 3) of foramina were assigned grades that did not appear in the original Lee grading system (grades B and D). The readers found no foramen that could not be described accurately with the updated grading system. Thus, an updated 6-point grading system for lumbar foraminal stenosis is reproducible and comprehensively describes lumbar foraminal stenosis as seen on high-resolution MRI.

Biglycan expression and its function in human ligamentum flavum.

Hypertrophy of the ligamentum flavum (LF) is a major cause of lumbar spinal stenosis (LSS), and the pathology involves disruption of elastic fibers, fibrosis with increased cellularity and collagens, and/or calcification. Previous studies have implicated the increased expression of the proteoglycan family in hypertrophied LF. Furthermore, the gene expression profile in a rabbit experimental model of LF hypertrophy revealed that biglycan (BGN) is upregulated in hypertrophied LF by mechanical stress. However, the expression and function of BGN in human LF has not been well elucidated. To investigate the involvement of BGN in the pathomechanism of human ligamentum hypertrophy, first we confirmed increased expression of BGN by immunohistochemistry in the extracellular matrix of hypertrophied LF of LSS patients compared to LF without hypertrophy. Experiments using primary cell cultures revealed that BGN promoted cell proliferation. Furthermore, BGN induces changes in cell morphology and promotes myofibroblastic differentiation and cell migration. These effects are observed for both cells from hypertrophied and non-hypertrophied LF. The present study revealed hyper-expression of BGN in hypertrophied LF and function of increased proteoglycan in LF cells. BGN may play a crucial role in the pathophysiology of LF hypertrophy through cell proliferation, myofibroblastic differentiation, and cell migration.

ADAM10 promotes the proliferation of ligamentum flavum cells by activating the PI3K/AKT pathway.

Ligamentum flavum hypertrophy (LFH) is an important cause of spinal canal stenosis and posterior longitudinal ligament ossification. Although a number of studies have focused on the mechanisms responsible for LFH, the cellular mechanisms remain poorly understood. The aim of the present study was to investigate the roles of differentially expressed genes (DEGs) in LFH, elucidate the mechanisms responsible for LFH and provide a potential therapeutic target for further studies. The GSE113212 dataset was downloaded from the Gene Expression Omnibus (GEO) database. The microarray data were analyzed and DEGs were obtained. Bioinformatics methods, such as Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment and protein­protein interaction (PPI) network analyses were used to obtain the key genes and signaling pathways. In addition, cells derived from hypertrophied ligamentum flavum were cultured, and the key genes and signaling pathways in ligamentum cells were identified through in vitro cell biology and molecular biology experiments. A total of 2,123 genes were screened as DEGs. Among these DEGs, 1,384 genes were upregulated and 739 genes were downregulated. The KEGG pathway analysis revealed that the DEGs were mainly enriched in the PI3K/AKT signaling pathway, and the PPI network analysis screened A disintegrin and metalloproteinase 10 (ADAM10) as a key gene. In vitro experimental verification revealed that ADAM10 promoted the proliferation of ligamentum flavum cells and led to the hypertrophy of the ligamentum by activating the PI3K/AKT pathway. On the whole, the in vitro experimental results suggested that ADAM10 promoted the proliferation of ligamentum flavum cells by activating the PI3K/AKT pathway, which may represent a pathogenic mechanism of LFH. The findings of the present study may provide a basis and direction for further studies on the cellular mechanisms of LFH and present a potential novel therapeutic target and clinical approach.

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.

The Role of JAK-STAT Signaling Activation in Hypertrophied Ligamentum Flavum.

BACKGROUND: Although previous studies have reported the expression of JAK1, STAT3, and phosphorylated STAT3 in hypertrophied ligamentum flavum (LF), the role of the Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling pathway in hypertrophied LF has not been fully elucidated. The aim of this study was to identify the important JAK/STAT gene expression patterns of the 3 main receptors involved in this pathway: interferon (IFN)-γ receptor (IFN-γR), IFN-α receptor (IFNAR), and interleukin (IL)-6 receptor (IL-6R). METHODS: The human LF specimens were obtained from 28 patients who underwent lumbar spine surgery for either degenerative lumbar canal stenosis (DLCS) (n = 28) or lumbar disc herniation (LDH) (n = 20). In this design, patients with LDH served as the control group. The degree of fibrosis was demonstrated by Masson's trichrome staining. The location and expression profiling of the JAK/STAT pathway were analyzed by quantitative real-time polymerase chain reaction and Western blotting. The thickness of the LF was measured with axial T1-weighted magnetic resonance imaging. RESULTS: The most severe fibrotic changes were on the dorsal side of the LF. IL-6 and IFN-I expression levels were significantly increased on the dorsal side of the LF. While expression levels of IL-6R and IFNAR on the dural and dorsal side were significantly higher in the DLCS samples, IFN-γR and endothelial epidermal growth factor receptor in LF samples showed a significant increase only on the dorsal side. JAK/STAT genes were significantly expressed, especially on the dorsal side. CONCLUSIONS: Our data suggest that IFNAR- and IL-6R-dependent JAK/STAT signaling pathways may be significant targets in drug development strategies for the treatment of LF hypertrophy.

Further characterization of a novel EP2 and EP3 receptor dual agonist, ONO-8055, on lower urinary tract function in normal and lumbar canal stenosis rats.

AIMS: To further explore the effects of a novel EP2 and EP3 dual agonist, ONO-8055, on detrusor contractility, we investigated the responses of bladder strips from sham and lumbar canal stenosis (LCS) rats to this agonist, its effects on lower urinary tract function in normal rats, and mRNA expression of EP2 and EP3 receptors in the sham and LCS rats. METHODS: The responses of bladder strips from sham and LCS rats to ONO-8055 were measured. The effects of ONO-8055 on LUT function of normal rats were investigated with awake cystometry and intraurethral perfusion pressure (Pura) measurements. The relative mRNA of bladder and urethral tissue of the sham and LCS rats was quantified using specific probes for EP1, EP2, EP3, and EP4 genes. RESULTS: Compared with the vehicle, the muscle tensions of both the sham and LCS rats were significantly increased after adding this agonist. On awake cystometry of normal rats, bladder capacity and Pura were decreased in the ONO-8055 groups, but a statistically significant difference in mean changes was demonstrated only between the vehicle group and the group receiving the highest dose. Compared with the sham rats, mRNA expressions of the four EP receptors in the lower urinary tract of the LCS rats did not show a statistically significant difference. CONCLUSIONS: This agonist did not augment bladder contractility or urethral relaxation in normal rats.

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.

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.

Increased advanced glycation end products in hypertrophied ligamentum flavum of diabetes mellitus patients.

BACKGROUND CONTEXT: Ligamentum flavum (LF) hypertrophy plays a dominant role in lumbar spinal stenosis (LSS). Although LSS prevalence is known to be higher in patients with diabetes mellitus (DM), the underlying pathomechanisms are not well understood. Abnormal advanced glycation end products (AGEs) formation occurs in DM and promotes tissue damage in various organs through degeneration and inflammation. PURPOSE: To analyze and compare LF histology focused on AGE status between control patients, LSS patients with DM, and LSS patients without DM. STUDY DESIGN/SETTING: Basic research study design utilizing human LF tissue for histologic analyses. PATIENT SAMPLE: LF tissue samples were collected from patients who underwent lumber decompression surgery for LSS in the author's institution. OUTCOME MEASURES: Quantitative visualization of Masson's Trichrome (MT) stains, and AGE immunohistochemistry (IHC) for the three groups. METHODS: Ten LF specimens from LSS patients with DM (DM group, mean age 71.4 years), 10 from LSS patients without DM (non-DM group, mean age 71.2 years), and 9 from patients with lumbar disc herniation or cauda equina tumor (control group, mean age 49.0 years) were harvested during surgery and histologically analyzed. Percentage of elastic fiber areas (%EF) was measured with MT staining, and the percentage of AGE immuno-positive areas (%AGEs) was measured with IHC. RESULTS: The average %EFs were 12.8 in the DM group, 17.1 in the non-DM group, and 24.9 in the control group. The decrease in the elastic fibers was significantly more in the DM group than in the non-DM (p<.01) and control groups (p<.001). Accumulation of AGEs was found mainly in the extracellular matrix in areas of elastic fiber disruption. The %AGEs were 18.3 in the DM group, 12.1 in the non-DM group, and 4.6 in the control group. These were significantly larger in the DM group than in the non-DM (p<.01) and control (p<.01) groups. The %AGEs also positively correlated with patient age (p<.01, R=0.47). CONCLUSIONS: Accumulation of AGEs is significantly greater in the LF of DM patients and correlates with patient age. AGEs may accelerate degeneration and hypertrophy of LF with age and may lead to higher prevalence of LSS in patients with DM. CLINICAL SIGNIFICANCE: The present results partly reveal the molecular mechanism of LF hypertrophy, suggesting that AGEs may be involved in the process of LF degeneration in the elderly and patients with DM.