The pathogenesis of intervertebral disk aging is poorly understood but is known to be associated with a variety of cellular and biochemical changes. As aging progresses, radial fissures of the annulus fibrosus progress outward from the NP. This process is associated with the activation of degradative enzymes, leading to weakening of the annulus fibrosus.1 Most extracellular matrix degradation is mediated by MMPs.2 Collagenase-3 (MMP-13) is expressed in chondrocytes of human adults3 and is overexpressed in chondrocytes of humans with osteoarthritis4 as well in affected disk tissues of rabbits and rats with experimentally induced disk degeneration.5–7 Thus, MMP-13 is a biological marker of, and may contribute to the pathogenesis of, intervertebral disk degeneration. However, the mechanisms that lead to upregulation of MMP-13 expression in degenerated disks are poorly understood.
The characteristic histologic change associated with disk aging or disk degeneration in humans is cell hypertrophy in the NP.8–10 Phenotypically, NP chondrocytes resemble cartilage chondrocytes.11 Previous in vitro research with cell lines and in vivo research in mice have shown that RUNX2 is required for chondrocyte hypertrophy12 and for MMP-13 expression in the hypertrophic region of bone growth plates.13,14 Runt-related transcription factor 2 expression is also induced in the articular cartilage of wild-type mice in early stages of osteoarthritis, and this induction occurs prior to MMP-13 expression,15 indicating that RUNX2 has an important role in the osteoarthritis disease process. Considering the phenotypical similarity between chondrocytes and NP cells, RUNX2 may also be implicated in the progression of intervertebral disk aging. However, the expression pattern of RUNX2 in intervertebral disks is unknown.
Human notochordal cells disappear by 10 years of age,16 but some nonhuman species appear to retain their notochordal cells for an extended period after attaining maturity. Chondrodystrophoid dogs, such as Dachshunds and Beagles, are among the few species that have intervertebral disk cell populations mimicking those found in adult humans.17 Dachshunds and Beagles are chondrodystrophoid dog breeds that become prone to disk disorders as early as adolescence.18 Because Dachshunds have the strongest tendency to develop intervertebral disk disease, it is easy to collect naturally occurring herniated disk specimens from dogs of this breed.18 Thus, the intervertebral disk material from these dogs may be useful for the investigation of mechanisms underlying disk aging in humans and chondrodystrophoid dogs.
A high degree of accuracy in detecting morphological changes in the spinal column attributable to aging and degeneration has been achieved with MRI. Investigators reported significant correlations of T2-weighted signal intensity with decreases in water and proteoglycan content,19,20 typical features of disk aging. Normal intervertebral disks have high signal intensity, whereas degenerating disks have lower signal intensity.20
The purpose of the study reported here was to investigate the relationship between RUNX2 expression patterns and disk aging in chondrodystrophoid dogs through examination of the spatial and temporal mRNA expression patterns of RUNX2 and MMP-13 (a biological marker of disk degeneration) in NP from intervertebral disks of healthy Beagles of various ages and from herniated intervertebral disks of Dachshunds. We also sought to compare signal intensity in T2-weighted MRI, which is a distinct marker for intervertebral disk aging, with RUNX2 protein expression.
Disk center–to–CSF T2-weighted signal intensity ratio
Rapid amplification of cDNA ends
Runt-related transcription factor 2
VISART, Toshiba Medical Systems Co, Tochigi, Japan.
Santa Cruz Biotechnology Inc, Santa Cruz, Calif.
RNA STAT 60, TEL-TEST Inc, Friendswood, Tex.
SuperScript III RT, Invitrogen Corp, Carlsbad, Calif.
GeneAmp PCR system 9700, PerkinElmer Cetus Corp, Norwalk, Conn.
3′-and 5′-Full RACE Core Set, Takara Bio Inc, Shiga, Japan.
ABI prism 3700 DNA sequencer, PerkinElmer Cetus Corp, Norwalk, Conn.
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