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molnan edited this page Aug 18, 2019 · 84 revisions

Welcome to the chemopreservation wiki!

About

This wiki is intended as a compendium of everything related to ultrastructural chemopreservation, that is, chemical preservation of either whole human beings or human brains at room temperature with enough quality to discern every ultrastructural detail of neurons that may play a role in encoding long-term memory and personality. In other words, techniques that can accomplish the goals of cryonics without the need for low temperatures.

Overview and basic concepts

Some concept definitions may help classify and compare different preservation techniques.

  • Ultrastructural preservation: any procedure that ensures long-term preservation of (at least) the human brain, including the ultrastructural details which encode long-term memory and personality according to widely accepted neuroscientific doctrine.

  • Ultrastructure-oriented preservation (UOP): any procedure that aims to achieve the goal of ultrastructural preservation, regardless of actual outcome. This concept is really useful when there's disagreement about the effectiveness of a particular procedure.

  • Uninterruptible cryogenic temperature UOP (AKA cryonics): a kind of UOP where preservation is achieved through cryogenic (or near-cryogenic) temperatures alone. The main focus is on minimal biochemical disruption, keeping the tissues as close to viability as possible. Since the tissues are not chemically fixed, and some ice crystal formation is sometimes allowed, this procedure assumes uninterrupted storage at cryogenic temperatures. It's worth emphasizing that long before autolysis and putrefaction become a concern, raising the temperature may lead to melting and mixing of damaged tissues, and even before that, to recrystallization. Both are potentially disastrous and must be avoided.

  • Interruptible cryogenic temperature UOP (in practice, vitrifixation): a kind of UOP where cryogenic temperatures are needed for long-term storage, but short periods at room temperature are considered non-fatal. In practice, some form of "vitrifixation". The idea is that chemical fixation protects the tissues from autolysis and putrefaction, and then high concentrations of cryoprotective agents provide robust vitrification at cryogenic temperatures for long-term storage. On one hand, this procedure doesn't seem very attractive, since it foregoes viability while still needing cryogenic storage, but there are some advantages. Compared to cryonics, we get tolerance to occasional, temporary failure. Since cryoprotectant concentrations are so high, the fluid is essentially unfreezable (it just vitrifies), so there's no risk of recrystallization. Compared to solid room-temperature UOP, the "embedding medium" can still include large amounts of water, so it's very gentle to cell mebranes and other delicate structures, and it can be easily introduced, hardened, melted and removed at safely low temperatures.

  • Aldehyde-Stabilized Cryopreservation (ASC): a particular vitrifixation protocol developed by McIntyre and Fahy. It consists of blood washout followed by perfusion fixation with glutaraldehyde, then perfusion with 65% w/v ethylene glycol as a cryoprotectant, and then vitrified storage at -135ºC. The fixation solution also includes some additives, like SDS to prevent tissue shrinkage and sodium azide to reduce mitochondrial swelling.

  • Interruptible refrigerated UOP (in practice, refrigerated liquid storage): a kind of UOP where refrigeration temperatures (1-4ºC) are used for long-term storage, but short periods at room temperature are considered non-fatal. In practice, this concept describes refrigerated liquid storage. It's assumed that subzero temperatures are not option (to avoid ice formation), otherwise the treatment would be identical to vitrifixation except for the actual storage temperature.

  • Ambient temperature UOP (acronym: ATUOP; can be called "ultrastructural chemopreservation" if not controversial): a kind of UOP where the specimen is stored without any form of active cooling, thereby passively acquiring the ambient temperature of the storage location. Durable, zero-maintenance, passive measures to avoid temperature peaks in hot regions, such as insulation and heat sinks would be acceptable. In practice, extreme heat should (and can easily) be avoided, so the "ambient temperature" won't be too far from the usual room temperature.

  • Liquid ambient temperature UOP (acronym: LATUOP; synonyms: ambient-temperature liquid storage, liquid chemopreservation; can be called "liquid ultrastructural chemopreservation" if not controversial): a kind of ATUOP where the specimen is preserved in a liquid bath. Its relevance is mainly historical.

  • Solid ambient temperature UOP (acronym: SATUOP; synonyms: solid chemopreservation; can be called "solid ultrastructural chemopreservation" if not controversial): a kind of ATUOP where the specimen is preserved in a fully solid state, thereby ensuring negligible diffusion damage during long-term storage.

The main emphasis of this wiki will be on solid chemopreservation, because it best fits the goal of affordable, zero-maintainance long-term storage while minimizing the risk of gradual information-destroying chemical damage.

Note: to the best of our knowledge, there's no need to define uninterruptible refrigerated UOP (no such protocol has been proposed), or to refine interruptible refrigerated UOP into solid and liquid forms (no solid form has been proposed).

Solid chemopreservation compared to cryonics and vitrifixation

If a SATUOP (solid chemopreservation) technique was developed that could serve as a drop-in replacement for cryonics (one which is applicable with acceptable quality in every situation where cryonics is an option), brain preservation would probably become a dramatically more affordable, reliable, convenient and popular practice. Room-temperature storage removes the need for:

  • long-term maintainance
  • special storage facilities
  • the continued existence of organizations responsible for those tasks

Of course, developing such a chemopreservation procedure is easier said than done.

Some casual discussion of the advantages, drawbacks and relevant issues:

Many techniques which are useful for cryopreservation are either directly applicable or easily adaptable to chemopreservation. Here's a nice summary of experimental cryopreservation strategies:

Vitrifixation has the advantage, among others, of being already validated in the form of ASC. Under ideal laboratory conditions, where both perfusion steps reach every region of the brain, the brain can be stored at room temperature for some time (it's not clear for how long) or even thawed and re-vitrified without substantial damage. In practice, though, some issues remain:

  • We don't know for how long the liquid-phase version would preserve the brain ultrastructure, so in practice we still need the maintainance associated with traditional cryonics, although with some tolerance to accidental thawing, which is indeed a big improvement.
  • The ideal lab conditions start with alive and healthy specimens, where perfusion quality is optimal. In contrast, cryonics patients must be declared legally dead before the procedure can start, and in many real-world cases, for a variety of reasons, PMIs (post-mortem intervals) end up being quite long. This means that some regions of the brain may end up poorly fixed and poorly cryoprotected, which would negate the advantages of ASC.
  • If current ASC protocols are indeed as sensitive to impaired perfusion as feared by their proponents (hence the BPF insistence on antemortem application), that means neither ASC nor any chemopreservation protocol with similarly stringent requirements can work as a drop-in replacement of traditional cryonics, only as an option for those lucky cases where PMIs are short enough.

State of the art

At this point there doesn't seem to be a peer-reviewed protocol for solid chemopreservation.

At least one cryonics organization (Oregon Cryonics) seems to offer a form of vitrifixation similar to ASC. Interestingly, they also offer liquid storage at a very affordable price. That said, it's a very new organization, we still need to confirm whether it's fully operational and those services are indeed available to the general public.

Relevant topics

Perfusion enhancement

It's well known in the context of cryonics that, even though neurons can withstand relatively long periods of ischemia, in practice every minute counts because perfusion becomes increasingly difficult:

  • Blood clots accumulate
  • Blood vessels collapse and become closed because of several factors, such as inflammation of the endothelium and external pressure caused by tissue edema
  • The blood-brain barrier breaks and then perfusion can flood and disrupt the brain parenchyma.

The mildest forms of impaired perfusion can be treated with medication according to clinical practice for stroke patients. The relevant concepts are reocclusion, no-reflow and hemorragic transformation. The effectiveness of those treatments is limited, though, and at some point capillaries and even larger blood vessels are simply too deteriorated to respond to medication.

Still, studies show that in some cases it's possible to fully restore brain circulation up to four hours post-mortem. This is especially relevant in that it suggests that perfusion fixation of good quality can be achieved after legal death, as is the usual practice in cryonics, without the need to seek a new legal framework where terminally ill patients undergo the procedure while still legally alive.

Cryonics procedures often perform minimal perfusion or ommit perfusion altogether when the PMI (post-mortem interval) is too long, going for a straight freeze instead. This is not an option for a chemopreservation protocol that involves storage at room temperature.

When perfusion becomes impossible or dangerous, the only alternative seems to be immersion fixation, which may or may not be enough (there are some encouraging results, but they are partially contradicted by other studies, so more data are needed).

Repairing the finest capillaries is way beyond current technology, but the idea is to restore access down to a certain blood vessel caliber, so that effective tissue thickness is no larger than a few millimeters. That would be enough to ensure tissues are fixed before autolysis becomes a big concern.

This restored access wouldn't provide circulation, because access to fine capillaries wouldn't be attempted. Instead, the perfusion fluid (eg a fixative solution) would either diffuse while staying at a given possitive pressure, or be subject to alternating flow, thereby entering and leaving the same blood vessels periodically.

It could be a similar procedure to catheter-directed thrombolysis but one that, besides removing blood clots, also opens up and reinforces not-too-small blood vessels via stents or some other means. It may also be desirable to increase their permeability, so that fixatives reach the brain parenchyma all along the blood vessel rather than only at the distal extremes. A laser could dig microscopic holes all allong, and then maybe a thin semi-permeable polymer layer can be added to perform a similar function as the blood-brain barrier, that is, to keep mannitol and other larger molecules inside the blood vessel while water is osmotically removed from the parenchyma. Then the fixative would penetrate quite easily due to its small size, or maybe the semi-permeable layer can be removed when edema is sufficiently reverted and mannitol is removed.

Immersion fixation

Immersion fixation of the human brain seems to be a standard practice, and neuronal ultrastructure seems to be preserved quite well, although probably not as well as with perfusion fixation. We have to figure out whether the fixation quality is enough. This is one of the key questions to answer in order to assess the viability of delayed post-mortem chemopreservation, because once the brain is fixed there's comparatively plenty of time for further treatments.

While glutaraldehyde can be used alone for perfusion fixation, its penetration rate is too low for immersion fixation. The usual choice is formaldehyde. It's also possible to combine the two, or or maybe use glyoxal instead, which seems to combine a penetration speed comparable to that of formaldehyde with the fixation quality comparable to that of glutaraldehyde, and with a relatively low toxicity.

Evidence for

  • 2018-02-20-Shatil-et-al: two human adult brains were stored in 10% formalin solution at room temperature for 1032 hours, doing CT scans at regular intervals and eventually inspecting them through optical microscopy. Neurons seemed to be fine, with myelin intact (see the section "Histological Validation of WM Integrity"). Sure, the resolution is not enough to evaluate the state of synapses, so we need more detailed studies.

  • 2014-04-11-Liu-Schumann: three right brain hemispheres from human adults were stored in 10% formalin at 4ºC for three years. Then samples were taken and stored in formalin for another 10 years. Some samples were also flash frozen. Preservation seems to be very good, and there are electron microscopy images available.

Evidence against

  • "Last year we decided to broaden our investigations to delayed chemical fixation and we have not been pleased at what we have observed so far. After 1.5 years of room temperature storage the delayed aldehyde fixed brains are falling apart and continue to decompose. In small animals one might imagine that such perfusion impairment could be overcome by immersing the brains in the fixative instead but human brains are simply too large. By the time that the fixative would have reached the core of the brain, extensive autolysis will have occurred." Source: In Praise of Cold

Vapor fixation

This treatment would be useful right after freeze-drying or a similar procedure that renders an unfixed, porous tissue. Formaldehyde would be the logical choice because of its volatility, but glutaraldehyde has also been tested successfully. Glyoxal would presumably work too. After this gaseous fixation step, it might be desirable to add a postfixation step for lipids, which would be also straightforward since osmium tetroxide is also volatile. This postfixation agent can't be used together with the aldehydes in the same step, because they react with each other. Vapor fixation with aldehydes and osmium tetroxide is well documented.

Freeze drying

Freeze drying would be an interesting option to look at if fixation can't be made fast enough. Freezing is comparatively quick. The downsides are:

  • Since we are assuming fixation isn't an option, we must also assume no cryoprotection, so there will be ice crystals
  • In fact, traditional freeze-drying techniques favor big, rather than small ice crystals, because otherwise the drying process takes too long.

We must, therefore, take a closer look at the damage inflicted during straight freezing, and to what extent it may lead to information loss.

Vacuum drying

Vacuum drying is a similar technique to freeze-drying, but it's done above the freezing point of water. The main advantages are the absence of ice crystals, and potentially shorter processing times. Since water evaporation cools the specimen, heat must be supplied to avoid freezing. If heat transfer isn't fast enough, the vacuum quality must be lowered (some pressure must be allowed) accordingly. In a vacuum there's no significant convective heat transfer, most is either conductive or radiative. Conductive heat transfer has the disadvantage of being a surface phenomenon. A "dry cake" tends to form in the contact zones, while the interior remains moist for much longer. This dry cake usually has a much lower heat conductivity, which makes matters worse. For that reason, radiative heat transfer is a frequent strategy. This is usually accomplished through microwave heating. Presumably infrarred light would be an improvement over conductive heating, but it can't transfer heat deep into thick specimens nearly as effectively as microwaves.

More information:

Freeze substitution

Freeze substitution is mainly a fixation technique for small samples in the context of electron microscopy, but there's also some literature about its use in whole organs.

The first step is to freeze/vitrify the tissue with the best quality available. Then the specimen is immersed at low temperatures (usually -80 or -90ºC, to avoid recrystalization) into a bath of acetone with osmium tetroxide (other organic solvents and fixatives are sometimes used). As the acetone slowly solubilizes and replaces the water, the osmium tetroxide ensures fixation of lipids and also proteins, nucleic acids and other relevant biomolecules. Then usually the organic solvent is replaced by an embedding medium such as thermosetting polymer resin. More information:

For small samples:

For whole organs:

High pressure treatments

High pressures (500-1000 bar) have some interesting effects that may be relevant.

  • They increase the penetration rate of formaldehyde (and possibly other fixatives).
  • They lower the melting point of water, so that tissues can be stored at lower temperatures without freezing, which gives more time for fixation.
  • This lower melting point is the basis of pressure-shift freezing, which is a very fast freezing technique. Small-crystal freezing or vitrification can be the first step of a chemopreservation protocol involving, for instance, freeze-substitution. Freeze-drying would be another option, but it can be very slow without large ice crystals.

According to a study on rat hearts, the best results are obtained at pressures no higher than 41 MPa (410 bar), which lowers the melting point to -4ºC. At 60 MPa (-6ºC) there was extensive edema, and at 78 MPa (-8ºC) there was significant morphological disruption.

More information:

Embedding

Thermosetting polymer embedding

This is a very common embedding technique in which a monomer infiltrates the specimen and it's later polymerized by raising the temperature (up to 37-60ºC, depending on the polymer). Usually, water is first replaced with an organic solvent (like acetone) which carries the monomer. This has the advantage of removing all the water (and therefore the long-term risk of biomolecule hydrolysis) but it may wash away cell membrane lipids unless an osmium tetroxide post-fixation step is added.

Mikula, Shawn & Denk, Winfried. (2015). High-resolution whole-brain staining for electron microscopic circuit reconstruction. Nature methods. 12. 10.1038/nmeth.3361.

Abstract:

Currently only electron microscopy provides the resolution necessary to reconstruct neuronal circuits completely and with single-synapse resolution. Because almost all behaviors rely on neural computations widely distributed throughout the brain, a reconstruction of brain-wide circuits-and, ultimately, the entire brain-is highly desirable. However, these reconstructions require the undivided brain to be prepared for electron microscopic observation. Here we describe a preparation, BROPA (brain-wide reduced-osmium staining with pyrogallol-mediated amplification), that results in the preservation and staining of ultrastructural details throughout the brain at a resolution necessary for tracing neuronal processes and identifying synaptic contacts between them. Using serial block-face electron microscopy (SBEM), we tested human annotator ability to follow neural 'wires' reliably and over long distances as well as the ability to detect synaptic contacts. Our results suggest that the BROPA method can produce a preparation suitable for the reconstruction of neural circuits spanning an entire mouse brain.

Note: Shawn Mikula passed away in 2018. It's unclear whether other researchers will continue his work on mouse brains. There's a study that uses a faster, modified version of BROPA (called fBROPA) on zebra fish brains, which are smaller.

On the other hand, there are water-soluble monomers such as GACH which don't damage membrane lipids. We need more data on possible downsides. Is water excluded from the resulting polymer matrix? Can the remaining water cause hydrolysis damage over time? Can it be gradually removed? How relevant is this for long-term preservation?

More information on GACH:

Paraffin embedding

In this procedure, tissue is dehydrated through a series of graded ethanol baths to displace the water, and then infiltrated with wax. The infiltrated tissues are then embedded into wax blocks. Once the tissue is embedded, it is stable for many years.

The most commonly used waxes for infiltration are the commercial paraffin waxes. A paraffin max is usually a mixture of straight chain or n-alkanes with a carbon chain length of between 20 and 40; the wax is a solid at room temperature but melts at temperatures up to about 65°C or 70°C. Paraffin wax can be purchased with melting points at different temperatures, the most common for histological use being about 56°C–58°C, At its melting point it tends to be slightly viscous, but this decreases as the temperature is increased. The traditional advice with paraffin wax is to use this about 2°C above its melting point. To decrease viscosity and improve infiltration of the tissue, technologists often increase the temperature to above 60°C or 65°C in practice to decrease viscosity.

Source: Paraffin Processing of Tissue

Sugar mixture embedding

Many sugars and sugar-alcohols as useful as lyoprotectants (substances that protect cell membranes and other structures against dehydration). By "sugar mixture embedding" we simply mean dehydration to a solid state (through air drying, vacuum drying or freeze-drying ) in the presence of enough lyoprotectant sugars and/or sugar-alchohols to provide both chemical protection and significant structural support against tissue shrinkage. At least the chemical protection part is amply discussed in the available literature.

More information:

PEG (macrogol) embedding

PEG embedding is an interesting alternative to paraffin embedding. Since PEG is water-soluble, there's no need to use solvents that may damage cell membranes. Also, for the same reason, residual water is presumably captured by the embedding medium instead of being excluded from it and kept close to the tissue, as it happens with paraffin. This might reduce the long-term risk of hydrolysis triggered by residual humidity. It has been tested on brain tissue with possitive results.

Slicing

Slicing is an extreme, radical option that should only be considered in two situations:

  • Even after every other trick has been tried, fixation is known to be too slow to prevent a dangerous level of autolysis, and the damage from freezing is considered worse than that from slicing, or freezing is rejected for some other reason
  • The whole brain has been stabilized (fixed or frozen) but it turns out to be too thick for room-temperature, solid-state chemopreservation to be practical (dehydration and/or embedding are self-limiting, or they are so exceedingly slow that they start to resemble long-term storage, with the associated increase in cost, risk and gradual deterioration).

Depending on the number of slicing, a significant amount of brain tissue is directly removed by the blade, and a much greater amount of neurons, while not directly dragged away, will inevitably suffer extreme damage in their dendrites and/or axon, which may accelerate other destructive processes.

The tool of choice is the vibratome. Slicing should probably be done at low temperatures (1-4ºC) to harden the tissue and delay autolysis.

Presumably (no directly relevant studies found so far), slow cutting in an aldehyde bath would ensure that only fixed portions of tissue are sectioned. Because fixative penetration slows down with depth (t=(d/K)^2 , where t is time, d is depth and K is a constant), fixation is relatively fast in the first few microns around the cutting edge, even if it's slow for the specimen as a whole.

More information:

Microscopy

Microscopy is an essential feedback tool to judge and compare the quality of each tissue preservation protocol. The ability to observe ultrastructural details becomes even more relevant when viability tests are no longer applicable, as is normally the case with chemopreservation.

The default choice, because of its high resolution combined with good speed and field width, seems to usually be some form of electron microscopy. Still, it would be highly desirable to test and validate alternatives to electron microscopy which are cheaper and more convenient, but sufficently precise to at least obtain preliminary results that can be useful in screening a wide variety of preservation protocols. For instance, optical microscopy is sometimes used to evaluate the preservation of myelin sheaths around axons.

FIB-SEM

FIB-SEM (Focused Ion Beam Scanning Electron Microscopy) seems to be the "gold standard". It was used by the BPF for its Brain Preservation Prize. In this technique a resin-embedded sample is destructively scanned one layer at a time, each layer removed through a focused beam of (usually gallium) ions. More information:

Expansion microscopy

Expansion microscopy (ExM) is a sample preparation tool for biological samples that allows investigators to identify small structures by expanding them using a polymer system.[1] The premise is to introduce a polymer network into cellular or tissue samples, and then physically expand that polymer network using chemical reactions to increase the size of the biological structures. Among other benefits, ExM allows those small structures to be imaged with a wider range of microscopy techniques. It was first proposed in a 2015 article by Fei Chen, Paul W. Tillberg, and Edward Boyden.[2] Current research allows for the expansion of samples up to 16x larger than their initial size.[3] This technique has been found useful in various laboratory settings, such as analyzing biological molecules. ExM allows researchers to use standard equipment in identifying small structures, but requires following of procedures in order ensure clear results.

Source: Wikipedia entry for "Expansion Microscopy"

More information:

Since expansion microscopy requires embedding in the expandible polymer (usually sodium polyacrylate), it doesn't seem compatible with irreversible embedding techniques like thermosetting polymer embedding.

Expansion microscopy has been combined with LSFM (Light sheet fluorescence microscopy) to obtain amazingly detailed 3D maps of the fruit fly brain.

More information: Thanks to rapid, 3D imaging, anyone can tour the fly brain

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