Ultrastructural preservation of a whole large mammal brain with a protocol compatible with human physician-assisted death

Building a high-fidelity computational model of the whole human brain will require preservation of the ultrastructure at the level of the entire organ, post-mortem. For such a model to reflect as closely as possible the brain in the living state, artifacts that arise during both the agonal phase and the postmortem interval will need to be minimized. This is potentially feasible if a terminally-ill patient donates their brain for research following physician-assisted death. In this paper, we modify a protocol for aldehyde-stabilized cryopreservation to make it compatible with physician-assisted death. We use pigs as a model, which resemble humans in cardiovascular and brain anatomy. Aldehyde-stabilized cryopreservation was designed to provide superior structural preservation of brains of any size, across all anatomical scales, compatible with diverse analytical assays and long-term storage without ultrastructural degradation. We demonstrate, with light microscopy and volume electron microscopy, that our brain preservation protocol results in connectomically traceable whole brains and propose an economically feasible storage modality that is expected to maintain stability of ultrastructure and macromolecules in the brain even for thousands of years. Most importantly, we establish that 14 min is the approximate length of the perfusability window—the time after the cardiac arrest during which blood washout needs to be initiated so that the brain ultrastructure is preserved.