Seven approaches for accelerating immersion fixation in brain banking

Immersion fixation of a human brain is fairly slow. One study found that it took an average of 32 days for single brain hemispheres immersion fixed in 10% formalin to be fully fixed (as proxied by achieving the minimum T2 value).

This means that fixative won't reach the tissue in the innermost regions of the brain until a substantial amount of tissue disintegration has already occurred.

Here are a few approaches to speed up immersion fixation in brain banking protocols. For each approach, I'm also going to list a rough, completely arbitrary estimated probability that they would each actually speed up the fixation process, as well as some potential downsides of each.

1) Cutting the brain into smaller tissue sections prior to immersion fixation. This approach is the most common approach already used to speed up immersion fixation. It relies on the obvious idea that if you directly expose more of the tissue to the fixative, the process of fixation will finish faster. I list it here for completeness.

Probability of speeding up immersion fixation: Already demonstrated.

Downsides: Damage at the cut interfaces, difficulty in inferring how cellular processes correspond between segments, mechanical deformation, technical difficulty in cutting fresh brain tissue in a precise manner.

2) Using higher concentrations of fixative. This makes biophysical sense according to Fick's law of diffusion, as a higher concentration gradient of fixative should increase its rate of diffusion into the tissue. One study found that 10% formalin led to a faster fixation rate in pig hearts, at least at the longest time interval studied (168 hours):

Holda et al 2017; PMID: 29337978; FBPS = Formaldehyde phosphate buffered solution

If 10% is faster than 2% or 4%, then 100% formalin would likely be faster than 10%.

Probability of speeding up immersion fixation with 50-100% compared to 10% formalin: 95%

Downsides: 100% formalin could produce more toxic fumes, it is likely more expensive, and it is not as easily accessible. It could also lead to more overfixation (e.g. antigen masking) of outer surface regions, although it theoretically could reach parity on this measure if a shorter amount of time were used for the fixation.

3) Using the cerebral ventricles as an additional source of fixative immersion.

If you can access the ventricles of the brain with a catheter or some other device, you could allow fixative to diffuse into the ventricles. This would allow for a substantially increased surface area from which fixatives can diffuse.

Because the cerebral ventricles are already there, using them allows for some of the advantages of the dissection approach without having to cut the brain tissue (other than the tissue damaged when placing the catheter(s) into the ventricles).

This approach can also be used when the brain is still inside of the skull, via the use of cranial shunts.

Access to the lateral ventricle is likely part of why immersion fixation is much faster after hemisecting the brain, which is already commonly done in brain banking protocols. 

Probability of speeding up immersion fixation: 50%. There are plenty of unknowns here. For example, are the ventricles already accessed through the cerebral aqueduct or canal when the brain is removed through the skull in standard immersion fixation? Do the ventricles collapse ex vivo or when the brain is taken out of the skull, rendering the approach much less effective? The uncertainty here should be attributed to my own ignorance of this literature, as other people are likely aware of the answers.

Downsides: Damage to parenchyma where the catheters are inserted, increased complexity of the procedure.

4) Using glyoxal or another fixative as a complementary agent. This is a pie-in-the-sky idea, but what about using a fixative other than formaldehyde? Glyoxal is one possibility. It has potential as an alternative fixative in terms of morphology preservation, and while it doesn't seem to be quite as efficient a crosslinker as glutaraldehyde, it might diffuse faster because it is smaller. I haven't been able to find good diffusion time measurements for glyoxal after a brief search. Glyoxal is also likely less toxic than formaldehyde.

Why use it at all if it likely diffuses slower than formaldehyde? It's not all about how quickly a fixative agent reaches the target tissue, but how efficiently it crosslinks once it gets there that is necessary to stop disintegration and stabilize the tissue. Glyoxal is the smallest dialdehyde so it might be a bit of a Goldilocks in the crosslinking efficiency vs diffusion speed trade-off. But, again, this is pie-in-the-sky and would need actual testing.

Probability of speeding up immersion fixation: 10% with glyoxal, 90% with some other fixative or combination of fixatives. It seems unlikely -- but possible -- that the first fixative ever used would just happen to be the best at immersion fixation of large tissue blocks.

Downsides: Other fixatives will likely be more expensive, less accessible, and cause artifacts that are harder to adjust for than the well-known ones caused by formaldehyde.

5) Ultrasound-enhanced delivery. Ultrasound has been shown to increase the speed of fixation in tissue blocks. One study found that ultrasound increased delivery speed of non-fixative chemicals (at the end of a catheter) by 2-3x. The mechanism is unknown, but could involve heat, which is already known to increase diffusion speed (not ideal, as this would also likely increase tissue degradation), and/or acoustic cavitation, a concept that I don't fully understand, but which can apparently speed liquid diffusion directly.

Probability of speeding up immersion fixation: 50%. I'd like to see these studies done on more brain tissue and for them to be replicated. However, they are pretty promising.

Downsides: Ultrasound might itself damage cellular morphology and/or biomolecules. However, considering that ultrasound has also been used in vivo, eg for opening the blood-brain barrier, it is unlikely to cause too much damage to tissue ex vivo, at least when using the right parameter settings.

6) Convection-enhanced delivery. This technique, which has primarily been used in neurosurgery, involves inserting catheters into brain parenchyma in order to help distribute chemicals such as chemotherapeutic agents. There's no reason why this couldn't be leveraged for brain banking as well.

Certain areas of the brain, perhaps the innermost ones that would otherwise take forever to be fixed, could be chosen to have small catheters inserted, allowing local delivery of fixative.

This would allow for an increase in the "effective surface area" of the fixative while minimizing damage due to sectioning and allowing the brain to remain intact.

Probability of speeding up immersion fixation: 99%. It's hard to see how using convective-enhanced delivery of fixatives with catheters inside of the brain parenchyma wouldn't speed up immersion fixation, but since I'm not aware of studies on it, there may be some technical difficulties that I'm not recognizing.

Downsides: Damage to the brain tissue from inserting the catheters, potential build-up of fluid pockets of fixative near the catheter tip that could damage nearby tissue if the infusion rate is too high, increased complexity, cost, and time for the procedure.

7) Shaking or stirring the fixative continuously (added 8/18/2019). This will increase the speed of fixative in an analogous way to convection-enhanced delivery: it delivers a pressure gradient, but instead of being inside of the tissue, it is at the surface.

The optimal rate of shaking or stirring is TBD and will depend on various factors specific to the experiment. Among other factors, there is likely a trade-off between such light shaking that it doesn't have an effect and such vigorous shaking that it will damage the brain tissue due to the translational motion, similar to a concussion.

Probability of speeding up immersion fixation: 99%. This approach makes perfect biophysical sense and it has already been shown to significantly increase fixation speed in freeze substitution. So it should very likely speed up the process of suprazero temperature fixation as well.

Downsides: Concussion-like damage to the brain, increased complexity, possible increase displacement of solutes within the brain.