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Rat tail static compression model mimics extracellular matrix metabolic imbalances of matrix metalloproteinases, aggrecanases, and tissue inhibitors of metalloproteinases in intervertebral disc degeneration.

ABSTRACT: INTRODUCTION: The longitudinal degradation mechanism of extracellular matrix (ECM) in the interbertebral disc remains unclear. Our objective was to elucidate catabolic and anabolic gene expression profiles and their balances in intervertebral disc degeneration using a static compression model. METHODS: Forty-eight 12-week-old male Sprague-Dawley rat tails were instrumented with an Ilizarov-type device with springs and loaded statically at 1.3 MPa for up to 56 days. Experimental loaded and distal-unloaded control discs were harvested and analyzed by real-time reverse transcription-polymerase chain reaction (PCR) messenger RNA quantification for catabolic genes [matrix metalloproteinase (MMP)-1a, MMP-2, MMP-3, MMP-7, MMP-9, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, and ADAMTS-5], anti-catabolic genes [tissue inhibitor of metalloproteinases (TIMP)-1, TIMP-2, and TIMP-3], ECM genes [aggrecan-1, collagen type 1-?1, and collagen type 2-?1], and pro-inflammatory cytokine genes [tumor necrosis factor (TNF)-?, interleukin (IL)-1?, IL-1?, and IL-6]. Immunohistochemistry for MMP-3, ADAMTS-4, ADAMTS-5, TIMP-1, TIMP-2, and TIMP-3 was performed to assess their protein expression level and distribution. The presence of MMP- and aggrecanase-cleaved aggrecan neoepitopes was similarly investigated to evaluate aggrecanolytic activity. RESULTS: Quantitative PCR demonstrated up-regulation of all MMPs and ADAMTS-4 but not ADAMTS-5. TIMP-1 and TIMP-2 were almost unchanged while TIMP-3 was down-regulated. Down-regulation of aggrecan-1 and collagen type 2-?1 and up-regulation of collagen type 1-?1 were observed. Despite TNF-? elevation, ILs developed little to no up-regulation. Immunohistochemistry showed, in the nucleus pulposus, the percentage of immunopositive cells of MMP-cleaved aggrecan neoepitope increased from 7 through 56 days with increased MMP-3 and decreased TIMP-1 and TIMP-2 immunopositivity. The percentage of immunopositive cells of aggrecanase-cleaved aggrecan neoepitope increased at 7 and 28 days only with decreased TIMP-3 immunopositivity. In the annulus fibrosus, MMP-cleaved aggrecan neoepitope presented much the same expression pattern. Aggrecanase-cleaved aggrecan neoepitope increased at 7 and 28 days only with increased ADAMTS-4 and ADAMTS-5 immunopositivity. CONCLUSIONS: This rat tail sustained static compression model mimics ECM metabolic imbalances of MMPs, aggrecanases, and TIMPs in human degenerative discs. A dominant imbalance of MMP-3/TIMP-1 and TIMP-2 relative to ADAMTS-4 and ADAMTS-5/TIMP-3 signifies an advanced stage of intervertebral disc degeneration.

SUBMITTER: Yurube T

PROVIDER: S-EPMC3446417 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Rat tail static compression model mimics extracellular matrix metabolic imbalances of matrix metalloproteinases, aggrecanases, and tissue inhibitors of metalloproteinases in intervertebral disc degeneration.

Yurube Takashi T Takada Toru T Suzuki Teppei T Kakutani Kenichiro K Maeno Koichiro K Doita Minoru M Kurosaka Masahiro M Nishida Kotaro K

Arthritis research & therapy 20120306 2

<h4>Introduction</h4>The longitudinal degradation mechanism of extracellular matrix (ECM) in the interbertebral disc remains unclear. Our objective was to elucidate catabolic and anabolic gene expression profiles and their balances in intervertebral disc degeneration using a static compression model.<h4>Methods</h4>Forty-eight 12-week-old male Sprague-Dawley rat tails were instrumented with an Ilizarov-type device with springs and loaded statically at 1.3 MPa for up to 56 days. Experimental load ...[more]

PMID: 22394620

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Project description:OBJECTIVE:To evaluate how well different magnitudes of compression-induced degenerative changes using a bent rat tail model simulated human lumbar lordosis. It has been shown that compression plays an important role in intervertebral disc degeneration (IDD). METHODS:Sprague-Dawley rats (n = 25) were instrumented with a special compressive apparatus that was used to bend the intervertebral disc between the 8th and the 10th caudal vertebral bodies using two Kirschner wires inserted percutaneously into the middle of two tail vertebrae. Then, rats were divided into five different static compression loads (control, sham, 1.8 N, 4.5 N, and 7.2 N). The degeneration of the discs was evaluated by magnetic resonance imaging (MRI), histology, gene expression of anabolism and catabolism after 2 weeks. We used the signal characteristics of the disc in T2-weighted MRI to reflect the changes caused by degeneration as this is the most relevant and clinically recognized way to assess IDD. Pfirrmann classification was used to classify disc images. The tail discs from C8-9 and C9-10 with their two adjacent half vertebrae were carefully cut out and decalcified. Then the sections were paraffin-embedded and cut into 5-?m sections by histotome. Finally, they were stained with Safranin O-Fast Green and hematoxylin, and hematoxylin and eosin, respectively. Images were taken using a microscope and staining and compression-induced changes were assessed by a Masuda's grading scale. The relative expression levels of mRNA encoding rat anabolic genes and catabolic genes were evaluated by real-time reverse transcription (RT)-polymerase chain reaction (PCR). The mRNA expression fold change of the target gene was calculated using the 2-??Ct method in the loaded and unloaded disc. RESULTS:As the loading magnitude increased, static compression produced a significantly progressive decrease in nucleus intensity on T2-weighted MRI, a decrease of aggrecan and Type II collagen, an increase in Matrix metallopeptidase-3 (MMP-3) and MMP-13 expressions, and a histomorphological degeneration. The sham group had a score of 1.4 ± 0.3, the 1.8 N group had a score of 2.4 ± 0.3, the 4.5 N group had a score of 3.2 ± 0.3, and the 7.2 N group had a score of 4.4 ± 0.3, which was based on the Pfirrmann classification score, in which the control group had a score of 1. These results demonstrated that the sham group was not significantly different from the control group. Histological analysis showed that in the loaded disc, the size of the nucleus was reduced and that the annular layer was disorganized. Based on the Masuda grading scale, scores were as follows: for the control group, 3.8 ± 0.35; sham, 4.2 ± 0.35; 1.8 N, 5.4 ± 0.35; 4.5 N, 7.6 ± 0.35; and 7.2 N, 10 ± 0.35. The gene expression was divided into the following: anabolic genes (aggrecan, collagen type1-?1, and collagen type2-?1) and catabolic genes (MMP-3 and MMP-13). Aggrecan and collagen type 2 were, respectively, downregulated from 0.42 ± 0.04 to 0.21 ± 0.04 and from 0.93 ± 0.06 to 0.17 ± 0.06 as the magnitude of compression increased, whereas collagen type 1 was significantly upregulated, from 2.49 ± 0.19 to 4.40 ± 0.19, when compared with the control group (from 1.8 to 7.2 N, P < 0.05). Catabolic genes MMP-3 and MMP-13 were significantly upregulated in all experimental groups (P < 0.05, MMP-3: from 1.46 ± 0.18 to 3.44 ± 0.18; MMP-13: from 1.19 ± 0.12 to 2.82 ± 0.13); however, MMP-13 exhibited no significant changes but tended to be upregulated when compared with the 1.8 N group with the 4.5 N group. CONCLUSIONS:Different stresses led to different processes of degenerative changes, the concave disc degenerating more severely as stress gradually increased.

Dataset Information

Rat tail static compression model mimics extracellular matrix metabolic imbalances of matrix metalloproteinases, aggrecanases, and tissue inhibitors of metalloproteinases in intervertebral disc degeneration.

Publications

Rat tail static compression model mimics extracellular matrix metabolic imbalances of matrix metalloproteinases, aggrecanases, and tissue inhibitors of metalloproteinases in intervertebral disc degeneration.

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