Evidence of White Matter R₂* Homogenization After Formalin Fixation

Disentangling intrinsic susceptibility-driven R₂* characteristics in white matter (WM) from fixation-related effects using in vivo, postmortem (PM) in situ, and formalin-fixed PM ex situ MRI.

DTI and multi-echo gradient-echo MRI were acquired in vivo, PM in situ and PM ex situ. PM cases included neurologically healthy individuals and patients with amyotrophic lateral sclerosis (ALS). Voxel-wise R₂* values were obtained using a mono-exponential fit and evaluated both as whole WM averages and according to WM fiber-to-field orientation. For the PM ex situ orientation-dependency analysis, PM ex situ R₂* data were registered to the corresponding in situ DTI acquisition. Averaged as well as orientation-dependent R₂* profiles were pooled across WM and compared across conditions.

Whole WM averaged and orientation-dependent R₂* values differed across in vivo, PM in situ, and PM ex situ conditions. No differences were observed between PM in situ healthy and PM in situ ALS cases. Compared with in vivo measurements, both PM conditions showed elevated whole WM averaged R₂* values and reduced orientation dependency, with the strongest R₂* anisotropy in vivo and nearly absent orientation dependency in the PM ex situ condition.

While orientation-dependent R₂* is reduced but preserved in unfixed PM in situ tissue, it is largely eliminated after prolonged formalin fixation without rehydration, likely due to microstructural reorganization and protein cross-linking. Collectively, these findings indicate that orientation-dependent R₂* measured in fixed tissue has limited translational validity, while underscoring its sensitivity to intact microstructural organization in vivo and in unfixed PM tissue.