All surgeries were performed by the 1st author at Kafkas University Hospital. LSCS and LDH were confirmed on sagittal and axial T1- and T2-weighted MRIs by the authors of this study and 1 skilled radiologist. MRIs were performed on a SIEMENS MR AEREA (1.5 Tesla) scanner.
2.2 Sample collection and measurement of LF thickness
The LF samples were obtained during surgical intervention. We obtained the whole layer of LF samples from all patients. Epidural fat was removed from the LF specimens, and the thickest point was measured using calipers to an accuracy of 0.01 mm. LF thickness was measured at 2 different points, and the largest thickness was used for analysis.
2.3 Histologic analysis
Half of each specimen was fixed in 4% neutral formalin and decalcified with 20% ethylene diamine tetraaceticacid for 4 to 6 weeks. Next, the specimen was embedded in paraffin for histologic analysis. Masson trichrome staining was used to evaluate the degree of fibrosis, and hematoxylin and eosin staining was used to evaluate elastin degradation. Histologic analysis was performed independently by 2 pathologists.
2.4 Masson trichrome staining
The severity of LF fibrosis was graded according to the guidelines by Sairyo et al: grade 0 indicates normal tissue, grade 1 indicates <25% fibrosis of the entire area, grade 2 indicates 25% to 50% fibrosis of the entire area, grade 3 indicates 50% to 75% fibrosis, and grade 4 indicates >75% fibrosis.
2.5 Hematoxylin and eosin staining
The degree of LF elastin degradation was also graded according to the guidelines by Sairyo et al: grade 0 indicates normal tissue showing no elastin degradation region, grade 1 indicates <25% elastin degradation of the entire area, grade 2 indicates 25% to 50% elastin degradation, grade 3 indicates 50% to 75% elastin degradation, and grade 4 indicates >75% elastin degradation.
2.6 Immunohistologic analysis
For immunohistologic staining, consecutive sections were cut on a microtome, deparaffinized in xylene, and rehydrated in alcohol solution. Next, 100 mg of LF tissue was homogenized with fetal bovine serum at 3000 RPM (Tissue-Tearor, 985370-04; BioSpec Products, Inc. Bartlesville, OK) and then lyzed in lysis buffer (50 mM Tris-HCl, pH 7.5, 10 mM ethylene diamine tetra acetic acid, 50 mM NaCl, 0.02% NaN3, 1% Nonidet P-40, 0.25 mM dithiothreitol). After centrifugation at 1500 RPM at 4°C for 30 minutes, the supernatant was obtained. For the analysis of each sample, 30 μg protein was loaded on to a 12% sodium dodecyl sulfate-polyacrylamide gel. The protein concentration of the lyzed LF tissue was determined using a bicinchoninic acid protein assay reagent kit (Pierce BCA protein assay kit no 23225, Thermo Fisher Scientific, Waltham, MA; Pierce Company). After electrophoresis, proteins were transferred to a polyvinylidene difluoride membrane (Millipore, St Quentin, Yvelines, France) for 2 hours at 150 V using transfer buffer. After blocking the nonspecific binding sites overnight, membranes were incubated with purified rabbit polyclonal antibody specific to catalase (rabbit anti-catalase; Cortex Biochem, CR2157RP, San Leandro, California). Mouse antibody specific to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control for protein loading for all samples (GAPDH antibody (0411): sc-47724). The results were visualized using enhanced chemiluminescence (Amersham Life Sciences, Piscataway, NJ). Blots were quantified using an Imaging Densitometer GF670 and molecular analysis software (Bio-Rad, SAFC Bioscience, Sigma-Aldrich) 3× for each sample, and the average was used as the final density. The density is presented as the mean ± standard deviation (arbitrary units).
2.7 Statistical analysis
Categorical variables are presented as the mean value and range. The Mann–Whitney U test was used to evaluate differences in the degree of LF elastin degradation, fibrosis, and mean optical density (OD) between the 2 groups. A P-value <.05 was considered statistically significant. All data were analyzed using SPSS version 12 statistical analysis software (SPSS Inc, Chicago, IL).
3.1 Demographic data
Participant characteristics are presented in Table 1. At the time of surgery, the average age in the LSCS group was 59.1 years (range, 34–79 years) and 44.05 years (range, 32–64 years) in the LDH group. The 2 groups did not differ significantly in terms of age (P = .850) or gender (P = .751).
3.2 Thickness of LF samples
The average LF thickness, measured during surgical intervention, was 5.99 mm (range, 4.5–7.02 mm) in the LSCS group and 2.95 mm (range, 1.98–3.2 mm) in the LDH group. The average LF thickness was significantly higher in the LSCS group (P = .004).
3.3 Elastin degradation and fibrosis of LF
In LDH (control) patients, the histologic images showed rich normal elastin fibers organized in a strictly parallel orderand regular arrangement (Fig. 3A). By contrast, in patients with LSCS, the grade of elastin degradation was very high with irregularly arranged elastic fibers shown by hematoxylin and eosin staining (Fig. 3B). The average grade of elastin degradation was significantly higher in specimens from patients with LSCS than from patients with LDH (3.04 ± 0.50 vs 0.79 ± 0.60, respectively, P = .007, Table 1).
In the LDH group, non fibrotic images were typically obtained (Fig. 3C). By contrast, large blue areas indicated massive fibrosis in images obtained from patients with LSCS (Fig. 3D). Further, the average grade of LF fibrosis was significantly higher in the LSCS group than in the LDH (control) group (3.01 ± 0.47 vs 0.66 ± 0.42, respectively, P = .009).
3.4 Catalase expression
Gelatin zymography of LF cell culture supernatant showed a sustained decrease in catalase activity in samples from patients with LSCS. In LF cell culture supernatant obtained from patients with LDH, higher catalase activity was observed (Fig. 4A) with standardization using GAPDH (Fig. 4B). The average OD was 61.80 ± 31.10 in the LSCS group and 152.80 ± 41.13 in the LDH group (Table 1, Fig. 5). The mean OD value was significantly lower in the LSCS group than in the LDH group (P = .009).
The LF covers the posterior and lateral walls of the spinal canal. As the LF thickens, the spinal canal begins to narrow and neural structures become trapped within the canal. In the present study, measurements made during surgery showed that the average LF thickness was significantly greater in patients with LSCS than in patients with LDH. We also demonstrated the presence of irregularly arranged, ruptured, swollen, and decreased elastic fibers as well as increased fibrosis in most areas of hypertrophied LF samples. Sairyo et al previously reported a positive relationship between LF thickness, loss of elasticity, and fibrosis score. Our results are consistent with those of Sairyo et al, and our inclusion of a control group strengthens these findings.
In addition, we sought to determine what biochemical factor might be responsible for histologic changes and LSCS. The catalase enzyme is known to decrease the secretion of structural proteins like collagen and angiopoietin in various connective tissues in the body. Previous studies have shown that overexpression of catalase prevents fibrosis.[14,15] Based on these findings, we hypothesized that a lack of catalase enzyme might play a role in LF hypertrophy. In the present study, we observed substantially lower catalase gene expression in the perivascular area of hypertrophic LF samples. Catalase is well known to protect cells against oxidative stress.[16,17] A previous study postulated that decreases in catalase activity cause abnormal epithelium repair. According to another study, epithelial damage is associated with functional deficiency of catalase during the development of fibrosis. Murthy et al reported that extracellular ROS increases matrix deposition and causes an increase in fibrosis in cells. Also ROS are known as reactive chemical entities that broadly participate in cellular signaling, metabolism, survival, and apoptosis, and thus ROS modulate many physiologic and pathologic processes including cell growth, fibrosis, contraction/dilation, and inflammation. Accordingly, our results suggest that a lack of catalase, which eliminates ROS, may be involved in the mechanism of LF hypertrophy and thus LSCS. This is the 1st study on the relationship between the catalase enzyme and LF hypertrophy and may suggest novel treatment strategies for LSCS.
The present study demonstrated that elastin degradation and increased fibrosis are closely related to LF hypertrophy, which is consistent with past studies. In addition, we reported a lack of catalase, which is an antioxidant enzyme, in patients with LSCS. We hope these novel findings have opened a door for future studies.
Conceptualization: Tayfun Çakir.
Data curation: Şeyho Cem Yücetaş.
Investigation: Tayfun Çakir.
Methodology: Şeyho Cem Yücetaş, Tayfun Çakir.
Resources: Tayfun Çakir.
Software: Tayfun Çakir.
Visualization: Tayfun Çakir.
Writing – original draft: Şeyho Cem Yücetaş, Tayfun Çakir.
Writing – review & editing: Tayfun Çakir.
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Keywords:Copyright © 2019 The Authors. Published by Wolters Kluwer Health, Inc. All rights reserved.
catalase; elastin degradation; fibrosis; ligamentum flavum hypertrophy; lumbar spinal canal stenosis