Update on storage at -20xC

[AndyMcKenzie]

We plan to convert our standard protocol to -20xC storage in the freezer room fairly soon, we expect within a year or two. I wanted to give a public update on this because many people have asked us about it.

Although I personally expect that fridge storage alone in formaldehyde/glutaraldehyde is likely sufficient to preserve the key aspects of the biomolecule-annotated connectome for decades, going to -20xC may be the more conservative option, because it may lead to fewer biochemical changes in the tissue over time.

There is no change to the initial perfusion fixation protocol. After fixation, we will do a graded ramp up of cryoprotective agent prior to placing the patients into the -20xC storage.

We are building off of a protocol in the literature that has been cited more than 1000 times and has been validated for decades. It is a bog standard tissue preservation protocol. The only addition we are adding is to do it across a whole brain, which is not as well validated. https://www.neuroscienceassociates.com/ ... hology.pdf

We have already validated once that this method does not cause microscopic changes to the fixed brain tissue after loading and unloading the cryoprotective agents. However, we want to replicate that result again. We don't want to cause any unnecessary damage and we want to be really sure of the method before we actually do the conversion on patients.

We will share more results when we have them.

[DetnPx]

Thank you, Andy,

What is your opinion on application of said protocol on a wholebody human patient through median sternotomy, applying said chemicals to the brain through the deceased human’s blood vessels (ideally quickly after MAID), and then preserving the whole body at -20xC?

Does this provide a substantial preservation difference to application and storage compared to just preserving the human brain?

Thank you for your potential insights.

[AndyMcKenzie]

Thank you for your interest and question.

Here are my thoughts on that protocol:

1. Based on the evidence I have seen to date, cannulating the internal carotid and vertebral arteries is a more efficient way of preserving the whole human brain. I believe it allows the procedure to start faster and it allows a larger volume of preservative chemicals to directly and rapidly enter the brain. That said, aortic cannulation and perfusion is also a reasonable approach.

2. A major issue with preserving the whole body based on CPA perfusion followed by -20xC storage is that the perfusion quality in the whole body is likely to be low, therefore there are some areas that will have ice crystals. Ice crystals are avoidable by preserving the whole body with chemical fixatives (perfusion to the extent possible, then immersion). This provides higher quality preservation in my opinion.

We will be offering whole body preservation as well, separate from the preservation of the brain. Whole body perfusion could be done via cannulation through the aorta or the carotids and vertebrals. We can do both. It is up to the person if they prefer aortic cannulation, but we recommend carotid and vertebral cannulation. If someone wants whole body preservation, then they could do carotid and vertebral cannulation for the brain, then still do perfusion of the rest of the body separately.

[jordansparks]

But I think the body would be stored at room temperature while the brain would be stored at freezer temperature.