Abstract
The intervertebral disc (IVD) is composed of three separate tissues with distinct origins and properties. Elucidating changes occurring in these tissues in response to injury or age is paramount to identify new therapies to better manage disc and spine degenerative conditions, including low back pain. Despite their small size and different mechanical load pattern compared to higher species, the use of mouse models represents a cost‐effective and powerful approach to better understand the formation, maintenance, and degeneration of the IVD. However, the isolation of the different compartments of the IVD is complicated by their diminutive size. Here, we describe a simple, step‐by‐step protocol for the isolation of the nucleus pulposus (NP) tissues that can then be processed for further analyses. Analysis from mouse NP tissues shows sufficient quantities of RNAs, purity of the NP fraction, and overall RNA quality for gene expression studies, and reveals no increase in expression of disc degeneration markers, including TNFa, IL1b, and Mmp1 up to 15 months of age in C57BL6 wildtype mice.
Highlights
The intervertebral disc (IVD) plays an important role in diffusing the various mechanical forces exerted on the spine during movement
More RNA was procured from lumbar nucleus pulposus (NP) (P = .007) and annulus fibrosus (AF)/cartilage endplates (CEP) (P = .006) when compared to thoracic counterparts (Figure 4C)
Bra and Cd24 expression was not different between NP and AF/CEP fractions, indicating significant contamination of NP RNA in AF/CEP samples, as these latter genes are not expressed in AF and CEPs (Figure 5C, D).[34]
Summary
The intervertebral disc (IVD) plays an important role in diffusing the various mechanical forces exerted on the spine during movement. Multiple studies support an anabolic and protective effects of NC cells on chondrocyte-like cells and their disappearance is associated with the onset of degenerative disc disease.[6–8]. Animal models have been utilized to better characterize healthy cell behavior and characteristics, disc formation and the degenerative process, despite the recognized morphologic differences between IVDs from small animals and humans.[11]. Studies in larger animals are aided in cell isolation by the gross morphological differences between the gelatinous NP and the fibrocartilaginous AF/CEP—these methods utilize gross dissection and further enzymatic digestion to ensure a high yield of cells.[12]. There is no consistent method for isolating cells of the different IVD compartments in mice for either cell culture or RNA expression (Table 1). We describe a new method to isolate pure NP RNAs from murine lumbar and thoracic IVDs, based on a simple centrifugation step
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