ASC consists of multiple steps which lock the brain's molecules into place and prevent them from decaying over time. First, ASC uses glutaraldehyde to almost instantly stabilize the brain against natural decay processes. By itself, glutaraldehyde is able to stop decay for at least several weeks, but we need more powerful stabilization to resist decay for centuries. In the second step, we use ethylene glycol to make it impossible for dangerous ice crystals to form in the brain no matter how cold it becomes. Then the brain can be safely cooled to -135°C (a temperature colder than anything which naturally occurs on Earth!) for long term storage. At -135°C, the molecules in the brain stop moving, forming a solid, "glassy" structure in a process called vitrification. Vitrification stops all decay processes and allows the brain to be stored for centuries with no degradation.

Glutaraldehyde is part of a family of aldehyde fixatives including formaldehyde, acrolein and glyoxal, all of which have been used in neuroscience to preserve brain tissue. All aldehyde fixatives have reactive aldehyde groups which can bind to proteins and create molecular crosslink bridges between them. Glutaraldehyde is a dialdehyde, which means it has two aldehyde groups, each of which is able to bind to proteins. Glutaraldehyde can also bind to itself to make chains. You can think of glutaraldehyde as a pair of molecular "handcuffs" — each aldehyde end is a "cuff", and the connecting carbon molecule is the "chain". When you expose brain tissue to glutaraldehyde, the glutaraldehyde rapidly binds to everything it can, transforming the brain from a soft, watery consistency to that of soft rubber. This process is called "fixation" and has been used for decades to preserve biological samples including brains, various organs, and even entire animals.

One of the reasons that brains deteriorate after death is that without a steady supply of energy and oxygen, the brain's metabolic processes go out of control, and the brain essentially digests itself. Glutaraldehyde stops all metabolic processes within seconds of contact and prevents runaway metabolism from damaging the brain's structure. The process of fixation also confers immense strength to the brain and makes it extremely resistant to changes in temperature, pH, and osmolarity. I've personally found that fixed brains can be stored for weeks at 4°C in a refrigerator with no structural degradation.

While glutaraldehyde is generally recognized as the "best" fixative for fixing biological tissue, it has one problem – it doesn't penetrate tissue very quickly. Glutaraldehyde penetrates tissues at around 0.75 millimeters / hour, which is far too slow to preserve a whole brain effectively simply by placing the brain in fixative solution. For example, it would take glutaraldehyde around 1 day to fully penetrate a whole rabbit brain. By that time, the core of the brain would have suffered severe degradation.

To get around glutaraldehyde's penetration problems, in ASC we deliver glutaraldehyde through the animal's blood vessels in a process called perfusion fixation. Every cell in a brain needs a steady supply of nutrients and oxygen, which are delivered by the bloodstream. Neurons are therefore never very far away from the nearest blood vessel; the average distance between a neuron and its closest capillary is less than 50 microns, meaning that you can fully deliver glutaraldehyde to every cell in the brain via perfusion fixation in less than 4 minutes.

Once the brain is fully fixed with glutaraldehyde, it is protected from decay for weeks, months, or perhaps even a year or two depending on how carefully it is stored. However, glutaraldehyde only binds proteins together and does not interact with lipids (a fatty substance which serves as the major structural component of cell membranes and myelin). Without further stabilization, these lipids will degrade over time. Fixed brains that have been stored for years often form a thick "film" at the top of their containers, made of lipids that have slowly migrated from their original positions in the brain due to the slow thermal action of water molecules.

In order to store the brain for longer than a century, we need a way to comprehensively stabilize all of the brain's structures against the random movement of water molecules. Extreme cold is one excellent way to do this, because for every 10°C decrease in temperature, the brain will last for twice as long without decay. This exponential increase in storage time adds up fast! For example, one week of cold storage storage at 4°C, a standard refrigerator temperature, is equal to 314 years at -135°C! Since glutaraldehyde allows brains to survive for much longer than a week at 4°C, if we could get the brain to -135°C, then the immense cold would allow us to store the brain for centuries without damage.

Unfortunately, a fixed brain is still mostly composed of water, and if you cool it down to -135°C, ice crystals will crush the brain's delicate neurons and synaptic connections, causing massive structural damage.

ASC avoids ice crystals in brains the same way you avoid them in your car during winter — with antifreeze. In ASC, we use a mixture of phosphate salts, ethylene glycol, and glutaraldehyde as a powerful cryoprotectant (antifreeze) solution. Ethylene glycol is the same chemical used in automotive antifreeze solutions and works by disrupting the hydrogen bonds between water molecules so that they can't link together to form ice crystals. The more ethylene glycol there is in a solution, the colder it can get before ice crystals form. However, once the concentration of ethylene glycol is high enough (around 55% weight / volume), ice crystals will never form, regardless of how cold the solution gets. Instead, as the solution gets colder it becomes more and more viscous until it becomes a vitreous (glass-like) solid, a process called vitrification.

There's no exact dividing line between a "solid" glass and an extremely viscous solution. Solutions that can vitrify become exponentially more viscous the colder they get. Eventually it might take years or even centuries for a very cold solution to flow at all! The glass in your windows is actually a vitrified solution of silicates, and if given enough time it would eventually "melt" into a puddle. (However, you would have to wait many trillions of years for this to happen in practice.) ASC's 65% ethylene glycol-based antifreeze solution becomes solid for all intents and purposes at -122°C (13°C warmer than the storage temperature of -135°C).

Ethylene glycol is normally very damaging to living brain tissue, causing immense dehydration and structural damage and generally "melting" the brain. Brain tissue fixed with glutaraldehyde is a different story; the glutaraldehyde fully protects the tissue from the destructive effects of ethylene glycol and allows us to completely cryoprotect the brain. Glutaraldehyde also buys us enough time to slowly add ethylene glycol to avoid mechanical disruption of the brain as water is gradually replaced with ethylene glycol.

In ASC, we add cryoprotectant slowly using a gradient former (Figure 4), a simple mechanical device which ensures that cryoprotectant is added slowly and uniformly over a course of 4 hours.

"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 frozen solid… This is not your father’s cryonics." — Dr. Kenneth Hayworth, BPF President

"I witnessed the infusion of a rabbit brain through its carotid arteries with a fixative agent called glutaraldehyde, which binds proteins together into a solid gel. The brain was then removed and saturated in ethylene glycol, a cryoprotective agent eliminating ice formation and allowing safe storage at −130 degrees C as a glasslike, inert solid. At that temperature, chemical reactions are so attenuated that it could be stored for millennia. If successful, would it be proof of concept?" — Michael Shermer, president of the American Skeptics Society

ASC is the first step towards a reliable nanoscale structural preservation protocol that can preserve all the information stored in a person's brain and body for hundreds of years. I've created a neuroscience research company called Nectome to do further research into ASC. I intend to create a robust preservation protocol that could be applied to humans and to clearly establish exactly what information is being preserved with ASC. If you're interested, please persue the following links to learn more!

ASC Frequently Asked Questions

Answers to common questions about neuroscience and cryobiology as related to ASC.

Cryobiology Paper

This is a peer-reviewed scientific account of the ASC protocol and early results obtained on rabbit and pig brains. The paper has been released open-access under a Creative Commons license.

Nectome

Neuroscience company dedicated to advanced structural brain preservation. I am one of the founders. Sign up for our mailing list if you want to stay informed about our progress.