Iodine-enhanced micro-CT imaging of soft tissue on the example of peripheral nerve regeneration

Abstract

Microcomputed tomography (µCT) is widely used for the study of mineralized tissues but a similar use for soft tissues is hindered by their low X-ray attenuation. This limitation can be overcome by the recent development of different 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. This optimization can be simplified 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 24h 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 fibroin tube can be visualized in 3D using this staining technique. This information helps in deciding the region of interest for histological imaging and provides a 3D context to histological findings. Correlating both imaging modalities has the potential to improve the understanding of the regenerative process.

Vascular Supply of the Human Spiral Ganglion: Novel Three-Dimensional Analysis Using Synchrotron Phase-Contrast Imaging and Histology

“…Lareida et al 15 , with a different synchrotron technique, and Müller et al 16 used staining with 1% OsO4 to visualize cellular structures, such as the organ of Corti, stria vascularis, membranous labyrinth, nerve fiber bundles, and even subcellular structures in ganglion cells. Although visualization is significantly improved, the process can lead to stain-induced shrinkage of the soft tissues 17 . Rau et al 18 obviated the use of staining through the use of phase-contrast imaging (PCI) to highlight soft tissues.…”

Development of a contrast‐enhanced micro computed tomography protocol for the oval squid ( Sepioteuthis lessoniana ) brain

“…Recently, Heimel et al () stated that double and stepwise staining could simplify the optimized staining of different tissues, prevented the overstaining, and minimalized the tissue shrinkage. Such staining methods would be effective for observing the complex structure of cephalopod brain.…”

Assaying three-dimensional cellular architecture using X-ray tomographic and correlated imaging approaches

“…The latter has become particularly prominent due to its ease of use, low toxicity, cost efficacy, reversibility and its systemic, yet differential staining of most tissues. Lugol's iodine ( Figure 3C) has thus been used to study a variety of samples, including murine embryos (76) and internal organs (77), neural tissues (78)(79)(80) and muscle (81)(82)(83)(84).…”