After fixation, maybe we should not store in liquid nitrogen

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jordansparks
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After fixation, maybe we should not store in liquid nitrogen

Post by jordansparks »

I've always had a little trouble explaining to people why we use fixative and then also cryopreserve. Fixative works very well, so what additional advantage are we getting from the liquid nitrogen? Everyone in the cryonics community has generally agreed that we need cryopreservation, so I've basically gone along with it. But that community might have a bit of bias, and I'm starting to question that position. Of course cryopreservation increases cost, complexity, and risk. But my real concern is that it might cause additional damage. For example, Alcor has posted images of a vitrified brain:
https://www.cryonicsarchive.org/library ... ied-brain/
That doesn't look like brain. They describe the problem as "dehydration artifacts". Yeah, for sure. When the brain gets dehydrated, it shrinks. This distorts all the detail. In theory, with mild shrinking, you could rehydrate and get all the normal detail back. In fact the brain images posted on our website have gone through that exact process:
https://oregoncryo.com/electronMicrographsVM1.html
They look a bit more normal than the Alcor images, but those brains were dehydrated and then rehydrated. This is not benign. I would characterize it as physical trauma. The reason Alcor is ok with this trauma is that ice crystals are worse. It's better to endure the dehydration than to risk ice crystals. But what if we didn't have to put up with either the dehydration or the ice crystal risk?
The arguments that I've heard against long term fixation are that damage would happen by just sitting there over the course of decades. But I have not seen evidence for this yet. Brain banks don't seem to be concerned with this issue. Their brains just sit there literally for decades. Most cryonics scientists don't even really consider this because they are more focused on cryopreservation without fixation. Fixation to them doesn't look like suspended animation, and it doesn't look revivable. They are flat out wrong about that of course, which I have covered extensively in other posts. There is a another group of scientists who sort of ignore cryonics and just talk instead about brain preservation with aldehyde. The size of that group is growing. One example is https://www.brainpreservation.org/.
We need some more science to help us decide which style of preservation causes the least amount of damage. The gold standard is getting images of tissue. Of course we can get stunning images of fixed brain tissue because fixation is simply part of the protocol for electron micrographs. We mostly need to measure tissue degradation over time. Since we can't wait decades for the results, we have to get creative. So we're working on that question. We also store all our fixed brains in refrigeration in order to help immobilize the remaining lipids. These are actually the molecules that I worry about because they are not locked in place by the fixative crosslinks. Instead, they are trapped in a web of proteins, but they very well might still migrate. The brain has a lot of lipids in it.
Another consideration is that there are different degrees of fixation. Most brains in brain banks are only moderately fixed with formalin, and then are transferred to a neutral saline storage medium with sodium azide for long term storage. The Brain Preservation group advocates for very aggressive fixation with glutaraldehyde. I would tend to agree that we probably want something stronger than formalin for our brains, but I'm unclear if the glutaraldehyde would distort the tissue. I'm guessing not since glutaraldehyde is always used when taking electron micrographs. We're looking into those issues as well.
My original question is still unanswered, but I feel like we're getting closer. There's a good chance that we might drop cryopreservation altogether for most cases. There would still be some where it would make sense such as previous straight freezes. I'm excited to see where this goes. If we generally drop cryopreservation, then we would rebrand under a new DBA name.
Darzin
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Re: After fixation, maybe we should not store in liquid nitrogen

Post by Darzin »

It's an interesting proposition, and definitely seems worth exploring. I do wonder if the added damage of cryopreservation might be considered worth it on a potential timeline of 1000s of years rather than decades. I suppose that's where more research is needed, but it does come down to a philosophical question in the end. How much damage is too much?

Regarding research do you and Apex Neuroscience have any plans to publish anything in the next five or so years? Either in a journal (though i'm not sure any would allow it) or just on this website.
jordansparks
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Re: After fixation, maybe we should not store in liquid nitrogen

Post by jordansparks »

Yes, we plan to regularly publish in mainstream science journals.

It might be time to rebrand away from "cryonics" right now. While we will always do some cryonics, the majority of our patients are chemically preserved instead. Perhaps the name of the company should reflect that majority. I also like the idea of distancing ourselves from the traditional cryonics companies. The goal really isn't to get the brain cold; the goal is to preserve the brain, and perhaps we should say so more clearly. I've reserved an appropriate domain and I'm getting very close to making the switch.

I'm remembering one argument that Mike Darwin always used in support of cryonics vs. chemical fixation. His basic claim was that fixative could never be guaranteed to reach 100% of the tissue, whereas cooling in liquid nitrogen could. But I think that's a bad claim. If the perfusion of fixative did not reach a certain area, then perfusion of cryoprotectant would also not reach that same area. Subzero cooling would then result in catastrophic ice damage rather than preservation. Preservation seems to require some chemical to reach the tissue, whether it's fixative or cryoprotectant. That's the real goal. Cooling is useless without first having achieved that. And if fixative reached the tissue, then the preservation has been accomplished. Cooling adds nothing except possibly better preservation over very long periods of time. But I'm increasingly skeptical that it even does that at all.
jordansparks
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Re: After fixation, maybe we should not store in liquid nitrogen

Post by jordansparks »

If we don't store in LN2, then we would at least store in refrigeration so that the lipids were more solid. So I started planning out a huge walk-in refrigerator where we could store many racks of small containers. It then occurred to us that maybe we should make it a walk-in freezer because that might provide slightly more stability. The downside is that each case would need to be ramped through cryoprotectant, which itself could be damaging. It's known that osmotic pressure can cause damage, so it might be complex to come up with a protocol that could be used safely and reliably. Complexity has a certain risk associated with it. Because we can always transition a patient from fridge to freezer, and from freezer to LN2, it seems like the walk-in fridge is still the best option for now. If we decide a freezer would be better, then we can add that later.
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