Open Access
Ex vivo, in situ perfusion protocol for human brain fixation compatible with microscopy, MRI techniques, and anatomical studies
(Frontiers Media, 2023) Insausti, Ricardo; Insausti Serrano, Ana María; Muñoz López, Mónica; Medina Lorenzo, Isidro; Arroyo-Jiménez, María del Mar; Marcos Rabal, Pilar; Rosa-Prieto, Carlos de la; Delgado González, José Carlos; Montón Etxeberria, Javier; Cebada Sánchez, Sandra; Raspeño-García, Juan Francisco; Iñiguez de Onzoño, María Mercedes; Molina Romero, Francisco Javier; Benavides-Piccione, Ruth; Tapia-González, Silvia; Wisse, Laura E. M.; Ravikumar, Sadhana; Wolk, David A.; DeFelipe, Javier; Yushkevich, Paul; Artacho Pérula, Emilio; Ciencias de la Salud; Osasun Zientziak
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.
Open Access
Postnatal development of NPY and somatostatin-28 peptidergic populations in the human angular bundle
(Frontiers Media, 2019) Cebada Sánchez, Sandra; Marcos Rabal, Pilar; Insausti Serrano, Ana María; Insausti, Ricardo; Ciencias de la Salud; Osasun Zientziak
The angular bundle is a white matter fiber fascicle, which runs longitudinally along the parahippocampal gyrus. It is best known for carrying fibers from the entorhinal cortex (EC) to the hippocampus through the perforant and alvear pathways, as well as for carrying hippocampal output to the neocortex, and distributing fibers to polysensory cortex. The angular bundle is already present prenatally at the beginning of the fetal period. Connections between the EC and the hippocampus are established by the 20th gestational week (gw). In the postnatal period, it shows increasing myelination. The angular bundle, as well as other white matter portions of gyral surfaces in the brain, presents interstitial neurons, a remnant of subplate neurons. Those interstitial neurons show neurochemical phenotypes both prenatally and postnatally, among which, neuropeptide Y (NPY) and Somatostatin-28 (SOM-28) peptidergic populations are noticeable, and accompany the fiber connections in the maturation of the hippocampal formation. We sought to investigate the topography of the postnatal distribution and relative density of neurons immunoreactive for NPY or SOM in the angular bundle along the rostrocaudal axis of the hippocampus. The study was carried out in 15 cases, ranging from 35 gws, up to 14 year old. All cases showed positive neurons showing a polygonal or spindle shaped morphology for both peptides, scattered throughout the angular bundle. The highest number of positive neurons appeared around birth and the ensuing weeks. Up to one and a half years, the density of both peptidergic populations decreased slightly. However, cases older than 2 years of age showed a substantial decrease in density of immunolabeled neurons, density that did not showed a minor decrease in density of positive neurons in cases older than 2 years. In addition, a topography from caudal to rostral levels of the angular bundle was detected at all ages. The functional significance of interstitial cells is unknown, but the existence of SOM and NPY peptidergic neurons, presumably inhibitory, in the white matter of the angular bundle, could contribute to the basic wiring of the hippocampal formation, through which autobiographical and spatial memories can begin to be stored in the infant brain.

Insausti Serrano, Ana María

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