Quantitative analysis of gene expression in a rabbit model of intervertebral disc degeneration by real-time polymerase chain reaction

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. 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. Gene expression analyses were performed to characterize a slowly progressive and reproducible animal model of IDD using real-time RT-PCR. Twelve rabbits underwent an annular stab with a 16-guage 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-1β ( IL-1β); 3) anabolic growth genes: bone morphogenic protein-2, and -7 ( BMP-2, -7), transforming growth factor-β1 ( TGF-β 1), 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. 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 preoper