The role of the PTHrP-IHH feedback loop in canine intervertebral disc degeneration

Abstract

Introduction - The vertebral column consists of vertebrae interconnected by intervertebral discs (IVDs). These IVDs consist of a nucleus pulposus (NP) surrounded by an annulus fibrosus (AF), and provide the flexibility and stability of the spine. IVD degeneration can cause IVD disease and is a relatively common reason for canine euthanasia. Current treatments for IVD degeneration and IVD disease aim to reduce the pain rather than repairing the IVD. Therefore, the development of regenerative treatments which do restore the biomechanical function is very important. To develop such a regenerative treatment, further knowledge of the pathogenesis of IVD degeneration and the pathways involved is required. The aim of this study was to determine the role of the Parathyroid Hormone-Related Peptide (PTHrP)-Indian Hedgehog (IHH) feedback loop in canine IVD degeneration. Methods - Canine IVDs with different Thompson scores were immunohistochemically stained for IHH. The mean percentage positive cells per Thompson grade was assessed in the NP and the correlation between the degenerative grades and IHH protein expression in the NP was determined. Also quantitative PCR for IHH, PTHrP and their receptors Smoothened (SMO), Patched (PTCH) and Parathyroid Hormone-receptor 1 (PTHR1) was performed on notochordal cells (NCs) that were cultured in monolayers and in clusters (culture day 0, 2, 4, 6, 8, 10, n = 8). To compare the in vitro qPCR results with the in vivo situation, cDNA from (non-digested, non-cultured) NC-rich NP tissue of six different NCD dogs was used as a control. Results - The mean percentage IHH positive cells decreased from Thompson grade I to II, where it was the lowest. From Thompson grade II to V this percentage increased. There was a significant positive correlation between IHH protein expression and IVD degeneration grade. In addition, NC gene expression of IHH, PTHrP and its receptor PTHR1 was decreased in the cultured NCs compared with the control group, while gene expression of the IHH receptors SMO and PTCH was increased compared with the control group. Conclusion - This study demonstrated a significant positive correlation between IHH protein expression and IVD degeneration grade, which may indicate that IHH stimulates IVD degeneration. Future studies are, however, needed to further elucidate the exact role of the PTHrP-IHH feedback loop in IVD degeneration and to determine if altering this pathway could be a point of engagement for the development of a regenerative treatment.