With the increased burden of low back pain (LBP) in our globally aging population there is a need to develop preclinical models of LBP that capture clinically relevant features of physiological aging, degeneration, and disability. Here we assess the validity of using a mouse model system for age-related LBP by characterizing aging mice for features of intervertebral disc (IVD) degeneration, molecular markers of peripheral sensitization, and behavioral signs of pain. Compared to three-month-old and one-year-old mice, two-year-old mice show features typical of IVD degeneration including loss of disc height, bulging, innervation and vascularization in the caudal lumbar IVDs. Aging is also associated with the loss of whole-body bone mineral density in both male and female mice, but not associated with percent lean mass or body fat. Additionally, two-year-old mice have an accumulation of TRPA1 channels and sodium channels Na v 1.8 and Na v 1.9 in the L4 and L5 lumbar dorsal root ganglia consistent with changes in nociceptive signaling. Lastly, the effect of age, sex, and weight on mobility, axial stretching and radiating pain measures was assessed in male and female mice ranging from two months to two years in a general linear model. The model revealed that regardless of sex or weight, increased age was a predictor of greater reluctance to perform axial stretching and sensitivity to cold, but not heat in mice.

Aging is a major risk factor of intervertebral disc degeneration and a leading cause of back pain. Pathological changes associated with disc degeneration include the absence of large, vacuolated and reticular‐shaped nucleus pulposus cells, and appearance of smaller cells nested in lacunae. These small nested cells are conventionally described as chondrocyte‐like cells; however, their origin in the intervertebral disc is unknown. Here, using a genetic mouse model and a fate mapping strategy, we have found that the chondrocyte‐like cells in degenerating intervertebral discs are, in fact, nucleus pulposus cells. With aging, the nucleus pulposus cells fuse their cell membranes to form the nested lacunae. Next, we characterized the expression of sonic hedgehog (SHH), crucial for the maintenance of nucleus pulposus cells, and found that as intervertebral discs age and degenerate, expression of SHH and its target Brachyury is gradually lost. The results indicate that the chondrocyte‐like phenotype represents a terminal stage of differentiation preceding loss of nucleus pulposus cells and disc collapse.

Intervertebral disc degeneration is the most significant, and least understood, cause of chronic back pain, affecting almost one in seven individuals at some point of time. Each intervertebral disc has three components; central nucleus pulposus (NP), concentric layers of annulus fibrosus (AF), and a pair of end plate (EP) that connects the disc to the vertebral bodies. Understanding the molecular and cellular basis of intervertebral disc growth, health, and aging will generate significant information for developing therapeutic approaches. Rapid and efficient preparations of homogeneous and pure cells are crucial for meaningful and rigorous downstream analysis at the cellular, molecular, and biochemical level. Cross-sample contamination may influence the interpretation of the results. In addition to altering gene expression, slow or delayed isolation procedures will also cause the degradation of cells and biomolecules that create a bias in the outcomes of the study. The mouse model system is extensively used to understand the intervertebral disc biology. Here we describe two protocols: (a) for efficient isolation of pure NP, AF, and EP cells from mouse lumbar intervertebral disc. We validated the purity of the NP and AF cells using Shh Cre/+ ; R26 mT/mG/+ dual-fluorescent reporter mice where all NP cells are GPF+ve, and by the sensitive approach of qPCR analysis using TaqMan probes for Shh, and Brachyury as NP-specific markers, Tenomodulin as AF-specific marker, and Osteocalcin as bonespecific marker. (b) For isolation of high-quality intact RNA with RIN of 9.3 to 10 from disc cells. These methods will be useful for the rigorous analysis of NP and AF cells, and improve our understanding of intervertebral disc biology.