Development and characterization of a novel non-surgical mouse model of intervertebral disc degeneration

Abstract

Purpose: In the spinal column, the purpose of the intervertebral discs (IVD) is to provide the spine with flexibility and load-bearing capacity. Each IVD is comprised of a highly hydrated nucleus pulposus (NP) core that is enclosed by the concentric collagenous fibers of the annulus fibrosus (AF). Maintenance of this composite structure and its biomechanical function requires proper activity of disc-resident cells, with balanced synthesis and degradation of macromolecular components. Despite that, our understanding of the role of senescence and cell death on the progression of disc degeneration is limited. IVD degeneration is one of the main contributors to low back pain and is known to precede several prevalent and debilitating spinal diseases, such as disc herniation and spondylosis. Although the causes of IVD degeneration are poorly understood, dysfunction and progressive decline in the number of resident cells have been found with aging and disease. Most animal models currently used to investigate the pathophysiology of IVD degeneration rely on surgical intervention, where invasiveness coupled with an inherent inflammatory response, limits our ability to mimic the pathogenesis of non-traumatic human disc degeneration. Hence, we sought to develop a non-surgical mouse model of IVD degeneration using genetically-induced targeted ablation of disc-resident cells that express aggrecan (Acan), the main proteoglycan component found in the extracellular matrix of the IVDs. Methods: We crossed heterozygous mice that express Cre recombinase under the control of the aggrecan gene promoter (AcanCreERT2) with mice that express diphtheria toxin (DTA) following Cre recombination (Gt(ROSA)DTA). The Acan+/Cre ;DTA+/- offspring received tamoxifen (4-OHT) interperitoneally for 5 (five) consecutive days to promote the targeted ablation of Acan-expressing cells. Animals were euthanized, and spines were harvested at 1-, 5-, 9- and 12-weeks post-DTA induction for histology. Sagittal sections were stained with Safranin-O/Fast-green and compared to littermate controls lacking the Cre allele (Acan+/+;DTA+/-) who received the same treatment, to allow a detailed spatiotemporal analysis of changes in the IVDs structure and composition. Long-term histological assessment was also performed at 7-months post-DTA induction, to investigate IVD degenerative changes with aging. Results: Histological analysis revealed mild degenerative changes of the IVDs by 5-weeks post-DTA induction in the Acan+/Cre:DTA mice, with the cells in the NP demonstrating a hypertrophic phenotype by 12-weeks, with few to no notochordal cells identified. Lumbar IVDs exhibited profound histological changes by 7-months post-DTA induction when compared to controls (Figure 1). These changes included decreased cellularity, loss of NP/AF borders, altered AF lamellar fiber structure, and disruption of NP matrix with loss of proteoglycan staining, and are typical characteristics of severe disc degeneration. Conclusions: Targeted cell depletion within the IVD resulted in progressive histological changes in the structure and composition of the NP, with clear degeneration of the AF compared to controls, reaching severe IVD degeneration by 7-months post-DTA induction. This novel mouse model of IVD degeneration does not require direct injury to the IVD; thus, it may contribute to further understanding the mechanisms underlying spontaneous and/or age-related degeneration of IVD, which more closely mimic the progression of human disease. Considering the structure-function relationship of this tissue, future studies should explore the mechanical behavior of IVDs to assess how the overall structural degeneration seen histologically translates into the biomechanical properties of the tissue.