Comparison of histological procedures and antigenicity of human post-mortem brains fixed with solutions used in gross anatomy laboratories.

Background: Brain banks provide small tissue samples to researchers, while gross anatomy laboratories could provide larger samples, including complete brains to neuroscientists. However, they are preserved with solutions appropriate for gross-dissection, different from the classic neutral-buffered formalin (NBF) used in brain banks. Our previous work in mice showed that two gross-anatomy laboratory solutions, a saturated-salt-solution (SSS) and an alcohol-formaldehyde-solution (AFS), preserve antigenicity of the main cellular markers (neurons, astrocytes, microglia, and myelin). Our goal is now to compare the quality of histology and antigenicity preservation of human brains fixed with NBF by immersion (practice of brain banks) vs. those fixed with a SSS and an AFS by whole body perfusion, practice of gross-anatomy laboratories. Methods: We used a convenience sample of 42 brains (31 males, 11 females; 25-90 years old) fixed with NBF (N = 12), SSS (N = 13), and AFS (N = 17). One cm3 tissue blocks were cut, cryoprotected, frozen and sliced into 40 µm sections. The four cell populations were labeled using immunohistochemistry (Neurons = neuronal-nuclei = NeuN, astrocytes = glial-fibrillary-acidic-protein = GFAP, microglia = ionized-calcium-binding-adaptor-molecule1 = Iba1 and oligodendrocytes = myelin-proteolipid-protein = PLP). We qualitatively assessed antigenicity and cell distribution, and compared the ease of manipulation of the sections, the microscopic tissue quality, and the quality of common histochemical stains (e.g., Cresyl violet, Luxol fast blue, etc.) across solutions. Results: Sections of SSS-fixed brains were more difficult to manipulate and showed poorer tissue quality than those from brains fixed with the other solutions. The four antigens were preserved, and cell labeling was more often homogeneous in AFS-fixed specimens. NeuN and GFAP were not always present in NBF and SSS samples. Some antigens were heterogeneously distributed in some specimens, independently of the fixative, but an antigen retrieval protocol successfully recovered them. Finally, the histochemical stains were of sufficient quality regardless of the fixative, although neurons were more often paler in SSS-fixed specimens. Conclusion: Antigenicity was preserved in human brains fixed with solutions used in human gross-anatomy (albeit the poorer quality of SSS-fixed specimens). For some specific variables, histology quality was superior in AFS-fixed brains. Furthermore, we show the feasibility of frequently used histochemical stains. These results are promising for neuroscientists interested in using brain specimens from anatomy laboratories.

Antigenicity is preserved with fixative solutions used in human gross anatomy: A mice brain immunohistochemistry study.

Background: Histology 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 methods: Twenty-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. Results: The 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. Conclusion: Solutions 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.

Laminar distribution of cortical projection neurons to the pulvinar: A comparative study in cats and mice.

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.

A novel ex vivo, in situ method to study the human brain through MRI and histology.

BACKGROUND: MRI-histology correlation studies of the ex vivo brain mostly employ fresh, extracted (ex situ) specimens, aldehyde fixed by immersion, which has several disadvantages for MRI scanning (e.g. deformation of the organ). A minority of studies are done ex vivo-in situ (unfixed brain), requiring an MRI scanner readily available within a few hours of the time of death. NEW METHOD: We propose a new technique, exploited by anatomists, for scanning the ex vivo brain: fixation by whole body perfusion, which implies fixation of the brain in situ. This allows scanning the brain surrounded by fluids, meninges, and skull, preserving the structural relationships of the brain in vivo. To evaluate the proposed method, five heads perfused-fixed with a saturated sodium chloride solution were employed. Three sequences were acquired on a 1.5 T MRI scanner: T1weighted, T2weighted-FLAIR, and Gradient-echo. Histology analysis included immunofluorescence for myelin basic protein and neuronal nuclei. RESULTS: All MRIs were successfully processed through a validated pipeline used with in vivo MRIs. All cases exhibited positive antigenicity for myelin and neuronal nuclei. COMPARISON WITH EXISTING METHODS: All scans registered to a standard neuroanatomical template in pseudo-Talairach space more accurately than an ex vivo-ex situ scan. The time interval to scan the ex vivo brain in situ was increased to at least 10 months. CONCLUSIONS: MRI and histology study of the ex vivo-in situ brain fixed by perfusion is an alternative approach that has important procedural and practical advantages over the two standard methods to study the ex vivo brain.