Quantitative analysis of gene expression in a rabbit model of intervertebral disc degeneration by real-time polymerase chain reaction.
BACKGROUND
Serial analysis of gene expression during the course of intervertebral disc degeneration (IDD) could elucidate valuable insight into pathophysiology and provide a basis for identification of potential targets for the development of novel cellular- and gene-based therapies. However, very few previous studies described the changes in gene expression through the process of IDD using a suitable animal model.


OBJECTIVE
To use a recently developed rabbit annular stab model and the technique of real-time reverse transcriptase-polymerase chain reaction (RT-PCR) to quantify the change in expression of key rabbit-specific mRNA sequences encoding for selected extracellular matrix (ECM) products, catabolic, anabolic, and anti-catabolic factors in normal and stabbed discs.


METHODS
Gene expression analyses were performed to characterize a slowly progressive and reproducible animal model of IDD using real-time RT-PCR.


METHODS
Twelve rabbits underwent an annular stab with a 16-gauge needle to the L2-L3, L3-L4, and L4-L5 discs, and three rabbits served as sham controls. Nucleus pulposus tissues were harvested from the stabbed discs at 3, 6, 12 and 24 weeks after confirmation of degenerative changes by magnetic resonance imaging (MRI) scan. Real-time RT-PCR was performed with the use of rabbit-specific primers for 1) extracellular matrix (ECM) component genes: collagen type Ia and IIa, and aggrecan; 2) catabolic genes: matrix metalloprotease-3 (MMP-3), inducible nitric oxide synthase (iNOS), and interleukin-1beta (IL-1beta); 3) anabolic growth genes: bone morphogenic protein-2, and -7 (BMP-2, -7), transforming growth factor-beta1 (TGF-beta1), and insulin-like growth factor-1 (IGF-1); and 4) anti-catabolic gene: tissue inhibitor of metalloprotease-1 (TIMP-1). These data were normalized to mRNA levels of glyceraldehyde phosphate dehydrogenase (GAPDH), a constitutively expressed gene.


RESULTS
The MRI images confirmed progressive decline in the nucleus pulposus area of high T2 signal and in the signal intensity of the stabbed discs over the 24-week study period consistent with IDD. The ECM components, aggrecan and collagen type IIa mRNA levels had decreased markedly by week 3 and never recovered, whereas type Ia collagen mRNA gradually increased throughout course of degeneration. BMP-2, BMP-7 and IGF-1 mRNA were relatively decreased from weeks 3 to 6 but then increased at weeks 12 and 24 to end at a level near the preoperative level. The TIMP-1 expression fell dramatically to approximately one tenth of the preoperative level by week 3 and remained low throughout the degenerative process. The remaining results, including those from TGF-beta1 and the catabolic genes (MMP-3, IL-1beta, iNOS) demonstrated a double peak characteristic. The gene expression increased by week 3, decreased to a low level at weeks 6 and 12 and then had a second, late peak at 24 weeks.


CONCLUSIONS
The gene expression profiles of ECM components and anabolic, catabolic, and anti-catabolic factors demonstrate many characteristics similar to the findings in human disc degeneration and suggest an inability of the intervertebral disc (IVD) to mount an early anabolic response to injury, thereby offering a possible explanation for the disc's lack of reparative capabilities. Catabolic genes are strongly up-regulated both early and late in degeneration, lending strong support to the hypothesis that an anabolic or catabolic imbalance plays a primary role in IDD. According to the resultant patterns, augmenting early production of BMP-2, BMP-7, IGF-1 or TIMP-1 by gene transfer techniques might possibly alter the progressive course of degeneration as seen in the stab model. The next step will be to transfer these therapeutic genes to regulate the biologic processes and ideally alter the progressive course of disc degeneration.
Sobajima
Satoshi
S
Ferguson Laboratory for Orthopaedic Research, Department of Orthopedic Surgery, University of Pittsburgh School of Medicine, 200 Lothrop Street PUH C-313, Pittsburgh, PA 15213, USA.
Shimer
Adam L
AL
Chadderdon
Robert C
RC
Kompel
John F
JF
Kim
Joseph S
JS
Gilbertson
Lars G
LG
Kang
James D
JD
D000818:Animals; D001363:Awards and Prizes; D001483:Base Sequence; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000235:genetics / Q000378:metabolism; D004195:Disease Models, Animal; D005109:Extracellular Matrix / Q000235:genetics* / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D005819:Genetic Markers; D007405:Intervertebral Disc Displacement / Q000175:diagnosis* / Q000235:genetics* / Q000601:surgery; D008279:Magnetic Resonance Imaging; D019278:Matrix Metalloproteinase 3 / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D011817:Rabbits; D012016:Reference Values; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism
C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D005819:Genetic Markers; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D019001:Nitric Oxide Synthase; D019278:Matrix Metalloproteinase 3