Characterization of the age-dependent intervertebral disc changes in rabbit by correlation between MRI, histology and gene expression

Abstract

Background: The present study was conducted to address whether the intervertebral disc of rabbit could be considered (i) as a valuable model to provide new insights into the tissue and cellular changes of Nucleus pulposus aging and (ii) as an appropriate tool to investigate the efficacy of Nucleus pulposus cell-based biotherapies.

Methods: Lumbar intervertebral disc from rabbits with increasing ages (1, 6 and 30 month-old) were compared by MRI and histological observation using Pfirrmann's grading and Boos' scoring respectively. The expression of transcripts (COL2A1, AGC1, COL1A1, MMP13, BMP2, MGP and p21) in Nucleus pulposus cells were analysed by quantitative real-time PCR.

Results: MRI analysis indicated an early age-dependent increase in the Pfirrmann's grading. Histological Boos' scoring was also increased. The analysis of transcript expression levels showed that COL2A1 and AGC1 were down-regulated as a function of age. Conversely, COL1A1, MMP-13, BMP-2, MGP and p21 were significantly up-regulated in the Nucleus pulposus cells of aged rabbit intervertebral disc.

Conclusions: Our study describes the consistency of the rabbit as a model of intervertebral disc changes as a function of age by correlating tissue alteration with cellular modification measured.

Figure 1

MRI images of the lumbar spine of rabbits with increasing ages. T2-weighted midsagittal images of (A) one-, (B) six- and (C) 30-month-old rabbits. Representative MRI images are shown.

Figure 2

Histological characterization of the Nucleus pulposus from IVD of rabbits with increasing ages. Histological sections of IVD from (A,D) one-, (B,E) six-, (C,F) 30-month-old rabbits were stained with (A, B, C) Haematoxilin Phloxin Safran and (D, E, F) alcian blue. Bar: 50 μm. Representative images are shown. (G) A Modified Boos's scoring was used to assess quantitatively the age-dependent tissue changes. *: p < 0.05 as compared to 1-month old rabbits; #: p < 0.05 as compared to 6-month old rabbits.

Figure 3

Phenotypic markers in Nucleus pulposus cells. Analysis of the expression levels of transcripts coding for the phenotypic markers (A) type II collagen, (B) Aggrecan, (C) type I collagen, (D) MMP-13, (E) BMP-2, (F) MGP and (G) p21 in Nucleus pulposus (NP) cells from IVD of rabbits with increasing ages. Messenger RNAs were purified from freshly isolated NP cells and analyzed by real-time PCR as described in the materials and methods section. Results are reported as fold change in gene expression related to one month old rabbits. *: p < 0.05 as compared to one-month-old rabbits; #: p < 0.05 as compared to six-month old rabbits.