MRI-histology correlation studies of the ex vivo brain mostly employ fresh, extracted (ex situ) specimens, aldehyde fixed by immersion. This method entails manipulation of the fresh brain during extraction, introducing several disadvantages: deformation of the specimen prior to MRI acquisition; introduction of air bubbles in the sulci, creating artifacts; and uneven or poor fixation of the deeper regions of the brain. We propose a new paradigm to scan the ex vivo brain, exploiting a technique used by anatomists: fixation by whole body perfusion, which implies fixation of the brain in situ. This allows scanning the brain surrounded by fluids, meninges, and skull, thus preserving the structural relationships of the brain in vivo and avoiding the disadvantages of ex situ scanning. Our aims were: 1) to assess whether months of in situ fixation resulted in a loss of fluid around the brain; 2) to evaluate whether in situ fixation modified antigenicity for myelin and neuron specific marker; 3) to assess whether in situ fixation improved the register of ex vivo brain images to standard neuroanatomical templates in pseudo-Talairach space for morphometry studies. Five head specimens fixed with a saturated sodium chloride solution (a non-standard fixative used in our anatomy laboratory for neurosurgical simulation) were employed. We acquired 3D T1-weighted (MPRAGE), 2D fluid-attenuated inversion recovery T2-weighted turbo spin echo (T2w-FLAIR), and 3D gradient-echo (3D-GRE) pulse sequences of all brains on a 1.5T MRI. After brain extraction, sections were processed for binding with myelin basic protein (MBP) and neuronal nuclei (NeuN) primary antibodies by immunofluorescence. This study showed that all but one specimen retained fluids in the subarachnoid and ventricular spaces. The specimen that lost fluid was the oldest one, with the longest interval between the time of death and the MRI scanning day being 403 days. All T1-weighted images were successfully processed through a validated pipeline used with in vivo MRIs. The pipeline did not require any modification to run on the ex vivo-in situ scans. All scans were successfully registered to the brain template, more accurately than an ex vivo-ex situ scan and exhibited positive antigenicity for MBP and NeuN. MRI and histology study of the ex vivo-in situ brain fixed by perfusion is feasible and allows for in situ MRI imaging for of at least 10 months post-mortem prior to histology analyses. Fluids around and inside the brain specimens and antigenicity for myelin and neurons were all well preserved.### Competing Interest Statement

Proof of concept of a novel ex vivo, in situ method for MRI and histological brain assessment