Introduction: With the increasing prevalence of chronic lower back pain (cLBP) and demand for pain management, there is a pressing need to better dissect the relationship between the structural and pathological changes in degenerated intervertebral discs (IVDs) and cLBP. We aim to determine the root cause of IVD pathologies and treat pain, focusing on developmental factors that regulate IVD formation and development. Previously, we showed that Sonic Hedgehog (SHH), a key developmental regulator, is expressed by young and healthy mouse nucleus pulposus (NP) cells of the intervertebral disc (IVD), and that SHH is essential for the maintenance of all components of the IVD in a young mouse. We also observed that age-related decline in SHH was associated with naturally occurring pathologies in the IVD, including pain and sensitization in aging mice. The current study aimed to test the role of SHH in the maintenance of IVDs in adult mice by developing novel conditional genetic mouse models and testing whether targeting Shh can accelerate IVD degeneration and associated pain at a molecular and behavioral level.
Methods: We generated Krt19CreERT/WT; Shhflx/flx mice, where, following tamoxifen induction, NP-specific conditional targeting of Shh can be achieved at any age (ShhcKO, n=29). The tamoxifen-treated Shhflx/flx (WT, n=23) littermates served as controls. All mice underwent behavioral assessments at nine months of age before tamoxifen administration (pre-ShhcKO), and at three- and six-months post-tamoxifen treatment (post-ShhcKO).Behavioral testing consisted of four tests: open field, tail suspension, acetone, capsaicin. At least two blinded observers recorded behavioral data. DEXA scans pre- and post-ShhcKO ensured body composition and IVD morphological comparability between groups. Cryosections from lumbar spine (L3-S1) were analyzed for morphometric, histopathological molecular changes in all components of the IVD. Immunofluorescence on IVD and lumbar DRG from the same mice assessed changes in the presence of Nav1.8, Nav1.9, TRPA1, CD31, and PGP9.5. Statistical analyses were conducted in GraphPad Prism version 10.
Results: Histomorphology and histopathological analysis showed significant degeneration in the IVDs of ShhcKO mice compared to WTs. ShhcKO mice exhibited substantial changes in behaviors indicative of pain compared to age-matched controls. DEXA measured no changes in the body composition between cohorts. Additionally, immunofluorescence highlighted increased innervation (PGP9.5) and vascularization (CD31) in the IVDs of experimental mice (ShhcKO), and an increased prevalence of nociceptive channels including Nav1.8, Nav1.9, and TRPA1 in their DRGs. Medication could alleviate neuropathic pain, but not the inflammatory pain in our model. Previously, we observed significantly higher expression of inflammatory factors by the IVD cells of the ShhcKO mice compared to controls.
Discussion: Our results demonstrate that age-related painful degenerative pathologies result from Shh loss. The results of the approach utilized in this study demonstrate the ability of the ShhcKO accelerated IVD degeneration model to portray naturally occurring aging. Further research is needed to determine whether rejuvenation of the IVD and treatment of its pathologies can alleviate pain at its origin without the risk of addiction.