Imbalanced Catabolic and Anabolic Gene Alterations
in a Rat Tail Compression Loading-Induced Disc Degeneration Model
Yurube T, +Nishida K, Suzuki T, Zhang Z, Yamamoto J, Kakutani K, Maeno K, Takada T, Kurosaka M, Doita M
+Kobe University Graduate School of Medicine, Department of Orthopaedic Surgery, Kobe, Japan
Senior author: [email protected]
INTRODUCTION
Low back pain is well-documented as a global health concern,
because of not only its high morbidity, but also high socioeconomic
costs involved in lost work hours and treatment. Although the
pathomechanism of low back pain is not clearly understood,
intervertebral disc degeneration is reported as a major cause. Disc
degeneration is a chronic remodeling process that is characterized
biochemically by the decreased cellularity and the degradation of
extracellular matrix (ECM). ECM production and activation is regulated
by the balance between degrading enzymes, matrix metalloproteinases
(MMPs) and aggrecanases, and their inhibitors, tissue inhibitors of
metalloproteinases (TIMPs). Although several authors reported the
imbalances of MMPs and aggrecanases relative to TIMPs in human
degenerated disc specimens1, further investigations using validated
animal models of disc degeneration are essential, because of difficulty in
reproducing systematic disc degeneration in human samples. The
objective of this study was to reveal comprehensive catabolic and
anabolic gene alterations throughout the disc degeneration process, using
a rat tail compression loading-induced disc degeneration model2.
METHODS
This experiment was approved by the Animal Care and Use
Committee of our institute. Forty-eight skeletally mature 12-week-old
male Sprague-Dawley rats were equipped with an Ilizarov-type device
with springs between the 8th and 10th coccygeal (C) vertebrae and loaded
for 0, 7, 28, or 56 days in static compression at 1.3 MPa2 (Figure.1).
Loaded (C8-9 and C9-10) and unloaded (C11-12 and C12-13) discs
were harvested. The total of twelve loaded or six distal-loaded discs in
six rats was used for evaluation in each time-point.
Radiographic evaluation: Radiographs were taken and the disc height
index (DHI) of each disc was calculated3. Changes in DHI of the loaded
discs were expressed as percent of baseline DHI and normalized to the
measured preoperative disc height (each n=12).
MRI evaluation: T2-weighted MRIs were also taken and classified into
the 5-grades using the Pfirrmann classification4 (each n=12).
Histomorphological evaluation: The loaded and unloaded discs were
excised from their respective vertebra-disc-vertebra and fixed. Sagittal
sections were stained with hematoxylin and eosin or safranin-O. These
sections were graded using the histological grading protocol established
by Masuda et al3 (each n=12). Additionally to confirm the progression of
imbalances of catabolism and anabolism, immunohistochemistry of
MMP-3 and TIMP-1, and ADAMTS-4 and TIMP-3, were performed.
Quantitative mRNA evaluation: Total RNAs were isolated from each
loaded C9-10 and unloaded C12-13 disc NP tissues. Using real-time RTPCR, the mRNA expressions of catabolic genes (MMP-1a, -2, -3, -7, -9,
-13, ADAMTS-4, and -5), anabolic genes (TIMP-1, -2, and -3), and
ECM genes [Aggrecan-1, Collagen type 1-α1 (Col-1), and type 2-α1
(Col-2)] were relatively quantified. GAPDH was used as an internal
control. These expression levels in the loaded discs were finally
normalized to the unloaded discs (each n=6).
Statistical analysis: One-way ANOVA and the Turkey-Kramer post-hoc
test were used for assessment. P-values were set at 0.05.
RESULTS
X-ray outcomes: Continuous disc height loss (Figure 2) and significant
decreases of %DHI were observed in the loaded discs.
MR outcomes: Lower NP intensities on T2-weighted images were
observed in the loaded condition than the unloaded (Figure 3) with
significant differences in the Pfirrmann grades.
Histological outcomes: Decreased NP cells with the matrix
condensations and ruptures of anulus fibrosus (Figure 4), and obviously
thicker staining for MMP-3 and ADAMTS-4 relative to TIMP-1 and -3
were detected in the loaded discs as the loading duration passed.
Quantitative mRNA outcomes:
[Catabolism] MMP-1a, -3, -7, -9, -13, and ADAMTS-4 showed
significant mRNA up-regulations from 7 days loading and significant
progressions as the loading duration increased, although a significant up-
regulation of MMP-2 was observed from 56 days. ADAMTS-5 however
did not any significant up-regulation during the loading period.
[Anabolism] TIMP-1 and -2 demonstrated no significant change but
increasing tendencies at the mRNA expression level. Meanwhile, TIMP3 mRNA expression was significantly down-regulated throughout the
loading duration.
[ECM] ECM genes demonstrated a significantly up-regulated expression
level of Col-1 at 56 days, while mRNA expression levels of Aggrecan1 and Col-2 were significantly down-regulated from 7 days loading.
DISCUSSION
Biological metabolism has a strong influence on the pathomechanism
of intervertebral disc degeneration; however, few animal model studies
have assessed them throughout the degenerative process. Although there
were several reports using a dynamic compression-loading induced disc
degeneration model, they were focused on acute and short-term
reactions. Accordingly, we investigated progressive alterations of
comprehensive catabolic and anabolic genes to the advanced stage of
disc degeneration in this study.
First, we assessed the validity of this rat tail disc degeneration model
by various evaluations. Progressive disc height loss in radiographs,
lower NP intensities on T2-weighted MRI, histological degeneration,
and ECM gene alterations such as an up-regulation of Col-1 and downregulations of Aggrecan-1 and Col-2, firmly validated this animal model
for disc degeneration research.
In this present study, progressive catabolic mRNA and proteindistributional up-regulations indicated the significant role of MMPs and
ADAMTS. On the other hand, TIMP-1 and -2 showed an insignificant
increase but there appeared to be a tendency toward up-regulation while
TIMP-3 showed significant down-regulation during the loading period.
These findings were consistent with the previous study of Le Maitre et
al.1. Although TIMP-1 and -2 might increase to counteract increased
MMP expressions, the existence of down-regulated TIMP-3 not
paralleled by up-regulation of ADAMTS-4 suggested that ADAMTS-4
could play an important role in disc tissue degradation, despite at least
no obvious up-regulation of another aggrecanase, ADAMTS-5,.
This rat tail model, used here to assess imbalances of catabolism and
anabolism, should prove useful for investigating the pathomechanism, as
well as developing molecular therapies for degenerative disc diseases5,6.
Figure 1. Rat instrumented with an Ilizarov-type device with springs
Figure 2. X-ray at 56 days loading
C8/9 C9/10
Figure 3. MRI at 56 days loading
C8/9
C9/10
C12/13
C12/13
Figure 4. Histology with hematoxylin and eosin at 56 days loading
C8/9
C9/10
C12/13
REFERENCES
1. Le Maitre et al. J Pathol 2004; 204: 47-54. 2. Lotz JC et al. Spine
2000; 25: 1477-83. 3. Masuda K et al. Spine 2006; 31: 742-54. 4.
Pfirrmann CWA et al. Spine 2001; 26: 1873-8. 5. Wertz K et al. J
Orthop Res 2009; 27: 1235-42. 5. Kakutani K et al. J Orhtop Res; 24:
1271-8. 6. Suzuki T et al. Eur Spine J; 18: 263-70.
Poster No. 1461 • 56th Annual Meeting of the Orthopaedic Research Society