OPTIMIZING GADOLINIUM-BASED CONTRAST AGENT PROTOCOLS FOR RELIABLE EX VIVO DIFFUSION TENSOR IMAGING IN AVIAN BRAINS

Understanding brain connectivity in non-mammalian species such as birds is essential for revealing the evolutionary foundations of cognition. Ex-vivo diffusion tensor imaging (DTI) offers high-resolution mapping of neural architecture, but its reliability depends on consistent tissue preparation and stable relaxation parameters. Gadolinium-based contrast agents (CAs) can enhance signal quality, however optimal application strategies for fixed avian brain tissue remain unclear. We evaluated different CA exposure strategies to establish an efficient ex-vivo DTI protocol for pigeon brains. Brains were assigned to four groups: [1] CA during perfusion, post-fixation, and washing; [2] during post-fixation and washing only; [3] during only washing; and [4] no CA (control). All samples were perfusion-fixed with 2% paraformaldehyde and after 5 days post-fixation were scanned with a 7T MRI at days 1, 7, 14, 23, and 70. Quantitative T1, T2, T2*, and DTI data were acquired using a single-shot EPI sequence (5 A0, 40 directions, b = 650 s/mm², TR/TE = 2100/30.5 ms). In Groups 1 and 2, T1, T2, and T2* increased until day 7 and stabilized by day 14, with no difference between them. In Group 3, relaxation values stabilized by day 14 as CA diffused into tissue, matching Groups 1–2. The control showed no T1 change but significantly higher T2 and T2* by day 70. CA exposure during washing alone thus yields stable, DTI-compatible relaxation parameters within two weeks, simplifying preparation while maintaining image quality. This minimal protocol enhances reproducibility in ex-vivo avian neuroimaging and supports standardized cross-species comparisons of brain connectivity.