Effects of perfusion fixation on whole-brain structural connectivity in marmoset: a diffusion MRI analysis

Ex vivo brain studies are essential in neuroscience research, yet the effects of perfusion fixation on structural connectivity remain poorly understood. We previously demonstrated that axial diffusivity is most sensitive to regional volume changes following fixation at the local tissue level. However, how these microstructural changes affect whole-brain network organization remains unclear. To investigate changes in whole-brain structural connectivity following perfusion fixation, advancing our understanding from local microstructural alterations to macroscopic network-level changes. Twelve common marmosets underwent 9.4T MRI scanning both in vivo and following perfusion fixation (ex vivo). Diffusion-weighted imaging was performed with optimized parameters for each condition. Whole-brain tractography was generated using constrained spherical deconvolution and anatomically-constrained tractography framework. Structural connectivity matrices were compared between conditions using the SIFT2 algorithm for quantitative assessment. We identified 799 connections showing significant differences between in vivo and ex vivo conditions (p < 0.05, Bonferroni corrected), 14.9% of all possible connections. The most affected regions included the temporopolar area (37 changed connections), hippocampal formation (32 connections), and mid-temporal area (29 connections). Brain regions with minimal volume changes showed fewer connectivity alterations, indicating that local tissue shrinkage predicts the degree of fixation-induced connectivity changes. Perfusion fixation substantially alters structural connectivity in a region-specific manner that correlates with local volume changes. Temporal and limbic regions are most vulnerable to fixation-induced connectivity alterations. These findings provide crucial reference data for interpreting ex vivo tractography studies and highlight the importance of considering fixation effects in comparative neuroimaging research.