High-Resolution 3-Dimensional Micro-CT Imaging of Intervertebral Discs Using a Novel Contrast Agent

Abstract

Background

The nucleus pulposus (NP) in the intervertebral disc (IVD) is the first structure to exhibit degenerative changes during IVD degeneration (IDD). Currently, micro-computed tomography (micro-CT) imaging of the NP is a limiting factor in detecting IDD at an early stage. While contrast-enhanced micro-CT has been investigated, an effective contrast agent for IVD has not been identified. This study investigates potassium iodide (KI) as an effective contrast agent for micro-CT-based IVD visualization across multiple animal models to study IDD.

Methods

We collected tails and spines from mice, rats, rabbits, sheep, and stained them with KI followed by micro-CT imaging. For IDD, we performed caudal annular needle puncture surgery (NPS) in age and sex-matched mice (n = 10) and stained with KI for imaging with micro-CT. For the aging model, we compared IVDs from old to young mice.

Results

Compared to unstained IVDs, KI effectively stained and visualized the 3D structure of the NP, exhibiting X-ray attenuation properties comparable to bone. KI contrast staining enabled accurate and reproducible quantification of IVD height and NP volume. The cross-sectional micro-CT images of NPS IVDs were indistinguishable from the histological findings of the same sample and showed similar degenerative changes in the NP. We also found that KI staining is reversible, and the tissue remains compatible with downstream histological processing and immunostaining. Notably, KI successfully stained the NP in decalcified tissue, offering an advantage for NP analysis by removing bone background in micro-CT scans. Additionally, we estimated that up to 15 sagittal sections, each 5 μm thick with 75 μm spacing, would be needed to fully assess IVD degeneration in mice.

Conclusions

This study demonstrated that KI can be used to positively stain NP in the intact tail or spine and provide qualitative and quantitative data without any adverse effects on the immune/histological processing of the samples.