Optimising neural contrast and minimising tissue shrinkage in dragon lizard brains: buffered Lugol's iodine versus phosphotungstic acid for contrast-enhanced μCT

Contrast-enhanced micro-computed tomography (CT) is increasingly used to study neuroanatomy without causing tissue damage. However, it still needs improvements for specific taxonomic groups and tissues. Researchers investigated how two common contrast agents, buffered Lugol's iodine and phosphotungstic acid (PTA), perform in visualizing the nervous system of the Jacky dragon lizard (Amphibolurus muricatus) via CT imaging.

They compared these agents based on staining time, contrast specificity, and brain tissue shrinkage at three concentrations (1.25%, 2.5%, and 5%).

Buffered Lugol's iodine proved superior to aqueous PTA, requiring less time to stain (as little as four days) and offering better differentiation between white and grey matter. PTA, on the other hand, needed longer incubation (about three weeks) and resulted in poorer differentiation between brain regions. Notably, buffered Lugol's iodine led to a higher estimate of brain-to-endocast occupancy, indicating significantly less tissue shrinkage.

In contrast, PTA-treated specimens showed a 13.3% reduction in occupancy and approximately 20% greater shrinkage of brain tissue compared to buffered Lugol's iodine.

These results show that buffered Lugol's iodine provides excellent contrast for detailed neuroanatomical analysis in three dimensions. It maintains the spatial relationships of anatomical structures and reduces artifacts, leading to a better match between the brain and its surrounding cavity. Practically, this study offers guidance on choosing the right contrast agent for future CT studies, highlighting that buffered Lugol's iodine is more suitable for fine-scale neuroanatomical, volumetric, and geometric morphometric analyses, while PTA is better for general soft-tissue or musculoskeletal visualisation.