Tissue clearing-enabled light sheet microscopy for three-dimensional mapping of innervation in the mouse intervertebral disc

Understanding the anatomy of healthy and degenerating intervertebral disc (IVD) innervation is crucial for deciphering the mechanisms of discogenic low back pain. While increased innervation has been linked to back pain, traditional 2D histology provides only a fragmented view of the delicate nerve networks in and around the disc. We leveraged tissue clearing, genetic labeling of sensory neurons, and the SPARC-null mouse model of progressive age-dependent disc degeneration to characterize lumbar disc innervation in 3D. SPARC-null mice were crossed with a Pirt-Cre;Ai14 reporter line to fluorescently label sensory neurons and were allowed to age to 12-months. This approach enables visualization of sensory innervation in healthy and degenerated discs without additional staining. Following perfusion and fixation, we obtained ex vivo MRI scans of the lumbar spinal column and graded lumbar IVDs based on signal intensity: white discs are highly hydrated and healthy, grey discs represent intermediate degeneration, and black discs indicate advanced degeneration. A subset of black discs showed visible herniations and were grouped as a separate phenotype. Discs were dissected, decalcified, and processed using the SmartBatch+ active clearing system (LifeCanvas) to preserve fluorescence and tissue integrity. Cleared discs were imaged using single-channel microscopy (RS-G4) and post-processed with a stitching and denoising pipeline implemented by the Minnesota Supercomputing Institute. This clearing approach enabled intact disc imaging with sufficient resolution to reveal sensory axonal networks in and around the discs and adjacent vertebrae. To validate the sensory selectivity of our endogenous Pirt-Cre;Ai14 tdTomato signal, we performed immunohistochemistry on sectioned spines from 12-month and 18-month old mice, staining for the pan-neuronal marker PGP9.5 and the nociceptive marker CGRP. This confirmatory analysis corroborates that our genetic labeling approach specifically captures sensory innervation patterns. Ongoing efforts will focus on computational quantification of innervation density and relationships between innervation and degeneration severity. This method enables high-resolution mapping of IVD innervation and may offer new insights into how sensory neurons may contribute to disc degeneration and the pathophysiology of back pain. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.