Ex vivo, in situ perfusion protocol for human brain fixation compatible with microscopy, MRI techniques, and anatomical studies

Abstract

We present a method for human brain fixation based on simultaneous perfusion of 4% paraformaldehyde through carotids after a flush with saline. The left carotid cannula is used to perfuse the body with 10% formalin, to allow further use of the body for anatomical research or teaching. The aim of our method is to develop a vascular fixation protocol for the human brain, by adapting protocols that are commonly used in experimental animal studies. We show that a variety of histological procedures can be carried out (cyto- and myeloarchitectonics, histochemistry, immunohistochemistry, intracellular cell injection, and electron microscopy). In addition, ex vivo, ex situ high-resolution MRI (9.4T) can be obtained in the same specimens. This procedure resulted in similar morphological features to those obtained by intravascular perfusion in experimental animals, provided that the postmortem interval was under 10 h for several of the techniques used and under 4 h in the case of intracellular injections and electron microscopy. The use of intravascular fixation of the brain inside the skull provides a fixed whole human brain, perfectly fitted to the skull, with negligible deformation compared to conventional techniques. Given this characteristic of ex vivo, in situ fixation, this procedure can probably be considered the most suitable one available for ex vivo MRI scans of the brain. We describe the compatibility of the method proposed for intravascular fixation of the human brain and fixation of the donor’s body for anatomical purposes. Thus, body donor programs can provide human brain tissue, while the remainder of the body can also be fixed for anatomical studies. Therefore, this method of human brain fixation through the carotid system optimizes the procurement of human brain tissue, allowing a greater understanding of human neurological diseases, while benefiting anatomy departments by making the remainder of the body available for teaching purposes.

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

“…At the same time, gross anatomy laboratories that use chemical fixation through perfusion of the whole-body cannot use this high concentration of NBF, since the bodies are typically used for dissection which would determine a long-term exposure to hazards for the users. However, a recent study of Insausti et al provided very good histology results following a carotid-perfusion method that enables to deliver formaldehyde to the brain, while allowing the injection of another fixative for the rest of the body ( Insausti et al, 2023 ). This could be an ideal solution in pursuing the utilization of 10% NBF for brain fixation by perfusion (avoiding the immersion confounds), while avoiding the hazard and inconvenience of high concentrations of formaldehyde for the dissection.…”

Postmortem imaging reveals patterns of medial temporal lobe vulnerability to tau pathology in Alzheimer’s disease

“…Before imaging, CNDR hemispheres were fixed in a 10% formalin solution for at least 30 days. HNL hemispheres were initially fixed in situ by perfusion with 8 L of 4% paraformaldehyde through both carotid arteries and then stored until processing in a cold room, submerged in 4% paraformaldehyde 63 . After fixation, the temporal lobe was extracted from every hemisphere and imaged overnight on a Varian 9.4 T animal scanner at a 200 x 200 x 200 μm 3 resolution using a multi-slice spin echo sequence.…”

Comparison of histological delineations of medial temporal lobe cortices by four independent neuroanatomy laboratories

“…At UCLM, fixation was performed by intracarotid perfusion with the brain in the skull (in situ; Insausti et al, 2023) before brain removal at autopsy with 4% paraformaldehyde in 0.1M phosphate buffer. At UPenn, brain specimens were fixed in 10% neutral buffered formalin after autopsy.…”