Background & Aims
Age-related intervertebral disc (IVD) degeneration and low back pain (LBP) are the top neurological conditions requiring most palliative care, which does not address the root cause. A better molecular understanding of the healthy adult disc and changes associated with its degeneration will provide clues into therapeutics developments for LBP aimed at treating the root cause. Each IVD has a central nucleus pulposus (NP) cells derived from the embryonic notochord and express Brachyury (mouse-Bra or T, human-TBXT), and is essential developmental regulator. We previously reported that NP cells of postnatal mice continue to express Bra. However, expression of Bra by NP cells declines with age and is associated with IVD degeneration. Moreover, the significance of Bra expression in the maintenance of adult discs is unknown. Goal of this study is to understand the role of Bra in maintenance of healthy IVD in adult mice.
Methods
To study the role of Bra in the maintenance of adult mouse IVD, we generated a Bra -flox allele and crossed it with tamoxifen-inducible conditional NP-specific driver line (Krt19CreERT) to generate Bra-cKO (Krt19CreERT; Bra-flox), and Bra-flox littermates (WT) served as controls. Tamoxifen was administered to 10-12-month mice, and the lumbar spine was dissected two weeks to six months later (post-Bra-cKO). The NP from lumbar IVDs in the two-week post-Bra-cKO was subjected to high-throughput bulk RNAseq. Pathway analysis was performed using Qiagen IPA. Validation of potential targets was performed by Multiplex qPCR was performed using TaqMan probes for the target gene along with B2m as a reference gene. Histological analysis and immunostaining of lumbar IVDs were analyzed to determine the effects of Bra-cKO on IVD pathology. Nikon microscope and NIS AR software captured and quantified images/data.
Results
RNAseq analysis of NP cells two weeks post-Bra-cKO showed a significant decline in extracellular matrix markers, TGFbeta, and Wnt signaling, and a significant upregulation of markers related to cell death, inflammatory pathways, and neurotropic factors compared to WT controls. Immunostaining and qPCR analysis validated the expression of critical targets. Moreover, lineage-tracing and histological analysis following three onwards showed dramatic changes in IVD histology, including phenotype like that of a two-year-old mouse IVD. These changes include bulging of the IVD, which in turn causes compression of spinal nerves, causing neuropathic pain. Hence, for the first time, we show the functional role of a notochordal marker in the maintenance of adult IVD.
Conclusions
Here, we present a novel conditional model of accelerated IVD degeneration, which will help study low back pain because of IVD degeneration. Moreover, as this conditional model is specific to the NP cells of the IVD, it will be helpful to evaluate the root cause of LBP and focus on therapeutics aimed at treating the IVD to prevent neurological symptoms and dependency on neuro-modulator painkillers.
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Presenting Author
Chitra Dahia
Poster Authors
Chitra Dahia
PhD
Weill Cornell Medicine, Hospital for Special Surgery
Lead Author
Topics
- Models: Chronic Pain - Neuropathic