Iodine-Enhanced Micro-CT Imaging of Soft Tissue on the Example of Peripheral Nerve Regeneration

Microcomputed tomography (ˆCT) is widely used for the study of mineralized tissues, but a similar use for soft tissues is hindered
by their low X-ray attenuation. is limitation can be overcome by the recent development of di‰erent staining techniques.
Staining with Lugol’s solution, a mixture of one part iodine and two parts potassium iodide in water, stands out among these
techniques for its low complexity and cost. Currently, Lugol staining is mostly used for anatomical examination of tissues. In the
present study, we seek to optimize the quality and reproducibility of the staining for ex vivo visualization of soft tissues in the
context of a peripheral nerve regeneration model in the rat. We show that the staining result not only depends on the concentration
of the staining solution but also on the amount of stain in relation to the tissue volume and composition, necessitating
careful adaptation of the staining protocol to the respective specimen tissue. is optimization can be simpli“ed by a stepwise
staining which we show to yield a similar result compared to staining in a single step. Lugol staining solution results in
concentration-dependent tissue shrinkage which can be minimized but not eliminated. We compared the shrinkage of tendon,
nerve, skeletal muscle, heart, brain, and kidney with six iterations of Lugol staining. 60 ml of 0.3% Lugol’s solution per cm3 of
tissue for 24 h yielded good results on the example of a peripheral nerve regeneration model, and we were able to show that the
regenerating nerve inside a silk “broin tube can be visualized in 3D using this staining technique. is information helps in
deciding the region of interest for histological imaging and provides a 3D context to histological "ndings. Correlating both
imaging modalities has the potential to improve the understanding of the regenerative process.