A cryogenically frozen rabbit brain has been returned from preservation in near-perfect condition. Researchers behind the breakthrough say there is no reason their technique could not be applied to larger mammals, including cows and primates, for long-term cryonic preservation.

The team at 21<sup>st Century Medicine has been awarded the Small Mammal Brain Preservation Prize for this achievement. Their study was published in the journal Cryobiology and shows how the neural circuits of an intact rabbit brain could be preserved in long-term storage using a preservation method they developed.

Kenneth Hayworth, president of the Brain Preservation Foundation, which runs the award, said: "Every neuron and synapse looks beautifully preserved across the entire brain. Simply amazing given that I held in my hand this very same brain when it was vitrified glassy solid... This is not your father's cryonics."

The researchers, led by Robert McIntyre, used a combination of chemical fixation and cryogenic cooling – which they call aldehyde-stabilised cryopreservation (ASC). In the study, they wrote: "To demonstrate the feasibility of ASC, we perfuse-fixed rabbit and pig brains with a glutaraldehyde-based fixative, then slowly perfused increasing concentrations of ethylene glycol over several hours in a manner similar to techniques used for whole organ cryopreservation."

Once a specific ethylene glycol level was reached, the brains were frozen at -135C for long-term storage. The brains were then rewarmed and evaluated for signs of damage.

"Preservation was uniformly excellent: processes were easily traceable and synapses were crisp in both species," they said. "ASC has many advantages over other brain-banking techniques: chemicals are delivered via perfusion, which enables easy scaling to brains of any size; vitrification ensures that the ultrastructure of the brain will not degrade even over very long storage times; and the cryoprotectant can be removed, yielding a perfusable aldehyde-preserved brain which is suitable for a wide variety of brain assays."

The researchers said they do not see any reason the technique cannot be used to preserve even larger brains, including cows, dogs and primates. However, they said human brains may prove more difficult because brain banks normally only receive them many hours after death.

rabbit brain cryogenic freezing
The rabbit brain preservation procedure BPF

"Furthermore, we do not see any simple way for ASC to be compatible with research protocols that require both fixed and unfixed tissue from the same brain; ASC delivers chemicals via perfusion and so must fix and cryopreserve the entire brain. Although we have emphasised the neurobiological applications of ASC here, our method is general and should be applicable to any organ system, or even to entire animals."

They added: "ASC promises to be a powerful new technique in the quest of connectomics researchers to unravel the mysteries of the mind. We also hope that ASC will inspire investigators in other fields to consider the possibility of cryobiological solutions to their problems."

Scientists working in cryonics have been searching for a way to put terminal patients into a state of long-term preservation in the hope that they could one day be woken up and cured by future medical and technological breakthroughs. Over the last 20 years, a number of breakthroughs have been made – specifically to stop ice forming on the brain. This is known as vitrification. However, while this prevents damage to cell structures, when it is applied to whole brains there is "dramatic shrinkage" of the tissues. Should McIntyre and his team's technique prove successful in larger mammals, this problem may be overcome.

In the longer term, scientists working on cryogenics hope to find a way to preserve not just the brain, but the memories it holds. "There is compelling preliminary evidence from the neuroscience literature that preserving a brain at the ultrastructure level... might also preserve the memories stored in that brain," the BPF said. "Now that whole brain preservation is a scientific reality, we look forward to a lively scientific debate over the exact level of preservation necessary to preserve memory."