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-α1, and collagen type 2-α1], and pro-inflammatory cytokine genes [tumor necrosis factor (TNF)-α, interleukin (IL)-1α, IL-1β, 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-α1 and up-regulation of collagen type 1-α1 were observed. Despite TNF-α 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.

Figure 1

Whole and close-up view of rat tail instrumented with an Ilizarov-type loading device.

Figure 2

Real-time reverse transcription-polymerase chain reaction gene expression profile. The messenger RNA (mRNA) expression of target gene normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is represented by fold change in the loaded relative to unloaded disc (control value = 1). *P < 0.05 when compared between loaded and unloaded conditions. ^†P < 0.05 when compared between different time points. (a) Relative mRNA expression at 0, 7, 28, and 56 days of: catabolic matrix metalloproteinase (MMP)-1a = 0.9, 1.6, 3.6, 6.1, respectively; MMP-2 = 1.2, 0.8, 3.4, 10.8, respectively; MMP-3 = 1.0, 5.0, 6.0, 9.8, respectively; MMP-7 = 1.0, 1.4, 1.9, 2.0, respectively; MMP-9 = 1.1, 1.3, 1.5, 1.5, respectively; MMP-13 = 1.0, 1.3, 1.5, 1.4, respectively; a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 = 1.0, 1.6, 1.9, 1.9, respectively; ADAMTS-5 = 1.0, 1.1, 1.1, 1.1, respectively. (b) Relative mRNA expression at 0, 7, 28, and 56 days of: anti-catabolic tissue inhibitor of metalloproteinases (TIMP)-1 = 1.0, 1.2, 1.4, 1.4, respectively; TIMP-2 = 0.9, 0.5, 1.2, 1.4, respectively; TIMP-3 = 1.0, 0.6, 0.7, 0.8, respectively. (c) Relative mRNA expression at 0, 7, 28, and 56 days of: extracellular matrix aggrecan-1 = 1.0, 0.3, 0.3, 0.5, respectively; collagen type 1-α1 = 0.9, 0.8, 1.6, 3.0, respectively; collagen type 2-α1 = 0.9, 0.6, 0.3, 0.2, respectively. (d) Relative mRNA expression at 0, 7, 28, and 56 days of: pro-inflammatory cytokine tumor necrosis factor (TNF)-α = 1.0, 1.9, 2.2, 2.0, respectively; interleukin (IL)-1α = 1.0, 0.7, 0.8, 0.9, respectively; IL-1β = 1.1, 1.1, 1.1, 1.2, respectively; IL-6 = 0.9, 1.0, 1.2, 1.2, respectively.

Figure 3

Immunohistochemical expression profile for matrix metalloproteinase (MMP)-3, tissue inhibitor of metalloproteinases (TIMP)-1, and TIMP-2. (a) Representative immunohistochemical loaded disc nucleus pulposus (NP) and annulus fibrosus (AF) sections for MMP-3, TIMP-1, TIMP-2, and negative control at 0, 7, 28, and 56 days of loading (bars: 50 μm). (b) Percentage of immunopositive cells of MMP-3 at 0, 7, 28, and 56 days in the loaded disc = NP: 44.1, 84.7, 79.0, 63.8, respectively, and AF: 19.5, 62.5, 60.4, 52.2, respectively; TIMP-1 = NP: 41.1, 36.7, 34.2, 9.4, respectively, and AF: 17.2, 20.8, 29.3, 11.9, respectively; TIMP-2 = NP: 47.2, 54.6, 27.6, 9.5, respectively, and AF: 15.8, 26.3, 25.3, 16.2, respectively. *P < 0.05 when compared between loaded and unloaded conditions. ^†P < 0.05 when compared between different time points.

Figure 4

Immunohistochemical expression profile for a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, ADAMTS-5, and tissue inhibitor of metalloproteinases (TIMP)-3. (a) Representative immunohistochemical loaded disc nucleus pulposus (NP) and annulus fibrosus (AF) sections for ADAMTS-4, ADAMTS-5, TIMP-3, and negative control at 0, 7, 28, and 56 days of loading (bars: 50 μm). (b) Percentage of immunopositive cells of ADAMTS-4 at 0, 7, 28, and 56 days in the loaded disc = NP: 45.4, 44.7, 51.1, 14.9, respectively, and AF: 17.1, 23.0, 52.4, 14.5, respectively; ADAMTS-5 = NP: 40.6, 45.7, 41.5, 8.7, respectively, and AF: 9.9, 24.3, 39.7, 8.8, respectively; TIMP-3 = NP: 40.5, 26.3, 12.1, 10.1, respectively, and AF: 13.0, 7.8, 5.5, 3.5, respectively. *P < 0.05 when compared between loaded and unloaded conditions. ^†P < 0.05 when compared between different time points.

Figure 5

Immunohistochemical expression profile for matrix metalloproteinase (MMP) and aggrecanase cleavage products of aggrecan. (a) Representative immunohistochemical loaded disc nucleus pulposus (NP) and annulus fibrosus (AF) sections for aggrecan neoepitopes generated by MMPs and aggrecanases and negative control at 0, 7, 28, and 56 days of loading (bars: 50 μm). (b) Percentage of immunopositive cells of MMP-cleaved aggrecan neoepitope at 0, 7, 28, and 56 days in the loaded disc = NP: 12.6, 71.2, 66.3, 61.5, respectively, and AF: 12.6, 52.6, 65.6, 58.5, respectively; aggrecanase-cleaved aggrecan neoepitope = NP: 15.9, 59.9, 75.2, 12.3, respectively, and AF: 18.7, 48.3, 59.7, 18.4, respectively. *P < 0.05 when compared between loaded and unloaded conditions. ^†P < 0.05 when compared between different time points.

Figure 6

Schematic illustration summarizing static compression loading-induced extracellular matrix metabolic imbalances of matrix metalloproteinases (MMPs), aggrecanases belonging to a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family, and tissue inhibitors of metalloproteinases (TIMPs) in the disc nucleus pulposus (NP) and annulus fibrosus (AF). A dominant imbalance of MMP-3/TIMP-1 and TIMP-2 relative to ADAMTS-4 and ADAMTS-5/TIMP-3 is an indication of an advanced stage of intervertebral disc degeneration.