Table 1.
Sequences of primers used for RT-PCR analysis.
Fig 1.
Pathological changes in LF tissue.
A: Hematoxylin-eosin (HE), picrosirius, trichrome, and Elastica-van Gieson (EVG) staining of LF in LSCS and control samples. Arrowheads indicate hyaline degeneration. Type 1 collagen appears red or orange and type 3 collagen appears green in color. Each scale bar, 100 μm. B: Average number of fibroblasts in the randomly selected field (×400). C, D: Average occupation of collagen fibers (C) and elastic fibers (D). E: Ratio of elastic and collagen fibers. F, G: Mean grade of LF fibrosis (F) and elastin degradation (G) by Park’s scoring systems. Lower grade (0 and 1) indicates less degeneration and grade 4 indicates severe degenerative change. H: Resorcin-fuchsin staining of LF samples in LSCS-fibrosis, LSCS-chondrogenic, and the representative control area. Arrowheads indicate elaunin-like fibers and arrows indicate oxytalan-like microfibers. Scale bar, 50 μm. I: Quantitative proteoglycan levels of LF tissues in LSCS and control. Data represent the mean ± SEM (n = 13 per group for histological assay; and n = 20 per group for proteoglycan assay, selected randomly). **P < 0.01 vs. control group.
Table 2.
Demographic data of the study population.
Fig 2.
MMP-2 and -9 expressions in LF tissue.
A, B: Comparison of MMP-2 (A) and MMP-9 (B) mRNA and protein expression in the LF of the LSCS and control groups. mRNA levels were normalized to 18S rRNA levels. The value in the control group was set as 1. C: Immunohistochemical analysis of MMP-2 and -9 expressions in LF tissue from patients with LSCS (left) and control subjects (right). The inset in the left panel for MMP-2 shows a high-magnification view of the area enclosed by the dashed line. Arrowheads indicate MMP-positive cells. Scale bar, 100 μm. D: Correlation between MMP-2 mRNA (left) and protein (right) expression and LF thickness. Data represent the mean ± SEM (LSCS, n = 31 and control, n = 21 for mRNA assay; and n = 14 per group for protein assay). **P < 0.01 vs. control group. N.S., not significant.
Fig 3.
Elastic fiber assay in LF tissues.
A: Correlation between MMP-2 mRNA expression and elastic fiber area. The minimum value of MMP-2 expression in analyzed samples was set to 1 (LSCS, n = 13 and control, n = 11). B: Elastin mRNA expression in LF tissue (LSCS, n = 31 and control, n = 21). Data represent the mean ± SEM. N.S., not significant.
Fig 4.
A: IL-6 mRNA expression in LF tissues from the LSCS (n = 31) and control (n = 21) groups normalized to 18S rRNA. Data represent the mean ± SEM. **P < 0.01 vs. control. B: Correlation between IL-6 and MMP-2 mRNA expression levels in LF tissues. C: Double immunofluorescence labeling of MMP-2 and IL-6. Nuclei were stained with DAPI. Scale bar, 100 μm. D: Immunohistochemical detection of p-STAT3. E: Quantitative analysis of data shown in panel D (n = 3). F: Immunohistochemical detection of p-ERK1/2 (upper), p-p38 (middle), and p-JNK (lower) in LF tissue from LSCS and control groups. Arrowheads indicate immunopositive cells. Scale bars, 150 μm. Regions of interest were selected from six sites (cranial, middle, and caudal sides of the dorsal and dural layers) in each sample. Images at 100× magnification were used for measurements, and the average number of p-STAT3-positive cells as a percentage of total number of cells was calculated. Data represent the mean ± SEM. **P < 0.01.
Fig 5.
MMP-2 expression and secretion induced by IL-6 in LF fibroblasts.
A: Changes in MMP-2 mRNA expression in LF fibroblasts (n = 3) following administration of IL-6 protein in the presence or absence of sIL-6Rα. MMP-2 expression in LF fibroblasts without IL-6/sIL-6Rα stimulation was set as 1. B, C: Changes in MMP-2 mRNA expression in LF fibroblasts (n = 3) in response to stimulation with IL-6/sIL-6Rα for 12 h at the indicated concentrations (B) and for the indicated times (C). D: MMP-2 protein concentration in the supernatants of culture with or without IL-6/sIL-6Rα stimulation for 24 h. E: Western blot analysis of STAT3, p-STAT3, ERK1/2, p-ERK1/2, p-38, p-p38, JNK, p-JNK, and actin levels in LF fibroblasts with or without IL-6/sIL-6Rα stimulation. Quantitative analysis of the ratio of p-STAT3/STAT3 (upper graph) and p-ERK1/2/ERK1/2 (lower graph) (n = 3) F: Nuclear translocation of p-STAT3 induced by IL-6 stimulation in LF fibroblasts. Nuclei were stained with DAPI. Scale bars, 100 μm. G, H: IL-6-induced MMP-2 mRNA expression (G) and secretion (H) with or without Stattic treatment (n = 3). Data represent the mean ± SEM. *P < 0.05, **P < 0.01, N.S., not significant.
Fig 6.
MMP-2 activation and elastolysis induced by IL-6 in LF fibroblasts.
A: Representative gelatin zymography analysis of LF fibroblasts in response to IL-6/sIL-6Rα stimulation for 24 h with or without Stattic. B: Quantitative analysis of data shown in panel A (n = 3). C: Concentration of soluble elastin following incubation for 6 h with the supernatant of IL-6-stimulated cultures with or without Stattic (n = 3). Data represent the mean ± SEM. **P < 0.01.