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BackgroundHistology remains the gold-standard to assess human brain biology, so ex vivo studies using tissue from brain banks are standard practice in neuroscientific research. However, a larger number of specimens could be obtained from gross anatomy laboratories. These specimens are fixed with solutions appropriate for dissections, but whether they also preserve brain tissue antigenicity is unclear. Therefore, we perfused mice brains with solutions used for human body preservation to assess and compare the tissue quality and antigenicity of the main cell populations.Materials and methodsTwenty-eight C57BL/6J mice were perfused with 4% formaldehyde (FAS, N = 9), salt-saturated solution (SSS, N = 9), and alcohol solution (AS, N = 10). The brains were cut into 40 μm sections for antigenicity analysis and were assessed by immunohistochemistry of four antigens: neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP astrocytes), ionized calcium-binding adaptor molecule 1 (Iba1-microglia), and myelin proteolipid protein (PLP). We compared the fixatives according to multiple variables: perfusion quality, ease of manipulation, tissue quality, immunohistochemistry quality, and antigenicity preservation.ResultsThe perfusion quality was better using FAS and worse using AS. The manipulation was very poor in SSS brains. FAS- and AS-fixed brains showed higher tissue and immunohistochemistry quality than the SSS brains. All antigens were readily observed in every specimen, regardless of the fixative solution.ConclusionSolutions designed to preserve specimens for human gross anatomy dissections also preserve tissue antigenicity in different brain cells. This offers opportunities for the use of human brains fixed in gross anatomy laboratories to assess normal or pathological conditions.

The cortical processing of visual information is thought to follow a hierarchical framework. This framework of connections between visual areas is based on the laminar patterns of direct feedforward and feedback cortico-cortical projections. However, this view ignores the cortico-thalamo-cortical projections to the pulvinar nucleus in the thalamus, which provides an alternative transthalamic information transfer between cortical areas. It was proposed that corticothalamic (CT) pathways follow a similar hierarchical pattern as cortico-cortical connections. Two main types of CT projections have been recognized: drivers and modulators. Drivers originate mainly in Layer 5 whereas modulators are from Layer 6. Little is known about the laminar distribution of these projections to the pulvinar across visual cortical areas. Here, we analyzed the distribution of CT neurons projecting to the lateral posterior (LP) thalamus in two species: cats and mice. Injections of the retrograde tracer fragment B of cholera toxin (CTb) were performed in the LP. The morphology and cortical laminar distribution of CTb-labeled neurons was assessed. In cats, neurons were mostly found in Layer 6 except in Area 17, where they were mostly in Layer 5. In contrast, CT neurons in mice were mostly located in Layer 6 across all areas. Thus, our results demonstrate that CT projections in mice do not follow the same organization as cats suggesting that the transthalamic pathways play distinct roles in these species.

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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 T1weighted (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.