Extracellular space preservation with immersion fixation of brain sections

Here’s a nice article from 2021: “Permeabilization-free en bloc immunohistochemistry for correlative microscopy” by Fulton and Briggman. A few thoughts:

1. The protocol helps to overcome an impediment to correlative en bloc EM and IHC by enabling the ultrastructural preservation of brain tissue and antibody penetration into relatively thick tissue sections, without the use of permeabilizing agents. This could help to facilitate the use of pre-embedding IHC in ultrastructural analysis techniques such as 3D EM.

They replicate the finding that permeabilization dramatically decreases ultrastructural quality, so should be avoided if possible:

https://elifesciences.org/articles/63392, Figure 1

They argue that a key way they were able to accomplish antibody penetration without permeabilization was via the preservation of the extracellular space (ECS).

2. The brains sections they used are still quite thin relative to those that are practical in neuropathology, at 300 um – 1 mm. In neuropathology, human brains are somewhat frequently sectioned at 5 mm, but even that is challenging and requires expert skill.

One could try to use a device such as a compresstome to help with the sectioning process. Here’s a video showing how the compresstome works on mouse brains. But it seems difficult to scale this to human brain sizes.

(If one were to achieve such thin sections in a high-fidelity way, you could theoretically cryopreserve them with vitrification or near-vitrification procedures and therefore avoid fixation altogether. Although, avoiding fixatives would also make room temperature preservation not currently possible.)

3. Another possible mechanism for why their protocol worked, that the authors did not discuss as far as I could tell, is that tissue decomposition during the immersion fixation process — which is slower than perfusion fixation — may itself cause membrane permeabilization. With a long enough time period of decomposition, cell membrane breakdown is an inevitable event, so the question is really whether the immersion fixation was slow enough to allow it to occur. My guess is that it was a contributing factor.

This may also help to explain why some epitopes are more accessible (eg Homer) than others (eg PSD-95). If a protein is a part of stronger gel-like networks, this gel-like network will likely break down slower during the decomposition process, and therefore be more difficult for antibodies to access without permeabilization.

4. Do we even need immersion fixation for ECS preservation? They cite Cragg 1980 as an example of a study that achieved ECS preservation using perfusion. It’s still not entirely clear to me why perfusion doesn’t usually achieve ECS preservation, but it seems like it probably depends on the osmotic concentration of the perfusate. Cragg 1980 is 30+ years old now; it would be ideal if it could be replicated and the phenomenon understood better.


Microwave irradiation as a brain banking tool

I love random reading old papers. They’re like treasure chests into secret knowledge that others might overlook. Here’s one: Guidotti et al 1974, “Focussed microwave radiation: a technique to minimize post mortem changes of cyclic nucleotides, dopa and choline and to preserve brain morphology”.

As a summary, they found that microwave irradiation in rat and mouse brains for 2 seconds led to the total inactivation of the enzymes involved in the regulation of numerous brain metabolites, such as cyclic AMP, cyclic GMP, choline acetyltransferase, and phosphodiesterase. As a result, it allowed for the accurate dissection of different brain nuclei for measurement of the concentrations of a variety of different metabolites in a variety of brain areas.

Rapid inactivation of enzyme activity; https://doi.org/10.1016/0028-3908(74)90061-6

The microwave irradiation method described by Guidotti et al. 1974 was relatively crude, as the authors only described microdissection of brain nuclei. It is unclear whether delicate cellular features such as synapses might also be preserved.

Interestingly, microwave irradiation produced extensive denaturation of proteins throughout the brain. Because since enzymes control metabolism, the inactivation of these enzymes can minimize changes in metabolism.

Microwave radiation could theoretically be combined with other brain preservation methods, such as fixation or cryopreservation, to minimize enzyme-driven autolysis during the procedure. A potential limitation of this method is that microwave heating is limited by the thermal conductivity of the tissue.

Human brain tissue can be effectively analyzed via electron microscopy at postmortem intervals up to 100 hours if the body is stored at cold temperature after death

I recently saw this interesting quote from Kay et al 2013 in their Nature Protocols article:

For tissue preparation, we have incorporated array tomography and EM preparations into routine brain bank collection. We have managed to conduct very effective EM studies on tissues retrieved from donors with long post mortem intervals, up to 100 hours. In our experience a key element in tissue preservation for ultrastructure analysis is post mortem cold storage of the cadaver, with cold storage in a mortuary of around 4–6°C significantly reducing structural degradation.


Synapses can be seen and analyzed via electron microscopy at 2-4 days postmortem in at least some cases

Many people will say that the brain decomposes completely within minutes after death. However, they usually don’t offer data when they make such claims.

My impression from reading the literature is that actual postmortem decomposition is slower than many people think.

Here’s an example from a recent article I read, Henstridge and colleagues 2015. They studied a brain tissue that they banked in part via immersion fixation in 4 % paraformaldehyde and 2.5 % glutaraldehyde in 0.1 M PB for 48 hours.

From Table 1, here is the pathoclinical information, including the postmortem interval. As you can see, some of the postmortem intervals prior to preservation are >3 days. It’s unclear to me if the body/brain was refrigerated during this postmortem interval, but it’s a likely possibility.

Table 1; doi: 10.1186/s40478-015-0232-0

As a side note, this is a fascinating data set that includes intelligence test scores at age 11. This allows the researchers to adjust for premorbid cognitive functioning in a robust way as they investigate the causes of age-related cognitive decline. 

After preparing the tissue for electron microscopy, they found that they were able to study synapses. Only a small percentage of the identified presynaptic and postsynaptic terminals were found to have degenerating profiles. This seems to have been attributed to antemortem Alzheimer’s disease, rather than postmortem decomposition.

Figure 13; 10.1186/s40478-015-0232-0

There are all sorts of confounds here. The study doesn’t seem to have been focused on identifying the limits of the postmortem interval in which this sort of study is still informative. They may have adjusted for the postmortem interval and presence of decomposed synapses in some sort of way. Et cetera.

But generally speaking, this data tells us that it’s reasonable to think that synapses might still be largely structurally intact even at 2-4 days postmortem interval. At least in some cases depending on the cause and circumstances of death. This is actual data, rather than pure speculation.

Five facts about decisions to donate to brain banks

From a nice systematic review on this topic by Meng-Jiun Penny Lin et al: 

  1. While most people know about organ donation, it seems that most people do not know about donating to postmortem tissue banks such as brain banks. One study found: “Although all participants were aware of organ donation for transplant, they were surprised that tissue could be donated for research. Nevertheless, once they understood the concept they were usually in favor of the idea. Although participants demonstrated a general lack of knowledge on donation for research, they were willing to learn more and viewed it as a good thing, with altruistic reasons often cited as a motive for donation.”
  2. Most brain tissue is donated by people with a neurobiological illness, but all brains are valuable. This is more so the case in the era of genomics, where tools such as PrediXcan allow researchers to impute the phenotypes of genotyped individuals by leveraging relationships from reference datasets. For such a reference mapping study, brains without significant neurobiological illness can be even more valuable, because it is less confounded by pathology and therefore can be more dispositive for early disease processes where most interventions are focused.
  3. When an early brain bank was established in 1961, donating one’s brain was seen as an “act of hope.” Lin et al note that altruism continues to be the primary motivator for brain donors: “The main reported motivation of participants across all 14 studies was desire to help others. A participant expressed the donation act as ‘a tiny step forward along with other people’.”
  4. Because so often the decision to donate is made by the next of kin, previous discussions between the donor and their family regarding the topic become critical. For example, one study found that “Almost a quarter (24%) commented that they had decided to donate because they were either aware that their deceased relative had wanted to be an organ donor, or believed it was something he or she would have wanted.” These conversations can also help alleviate donor’s anxieties that their wishes will not be followed.
  5. One study found that there was an inverse relationship between how long after death the conversation about donation occurred and how likely the next of kin was to consent (p = 0.01).

    Screen Shot 2019-09-21 at 8.35.37 AM
    Garrick et al 2009, 10.1007/s10561-009-9136-1

    It was a relatively small study and the finding needs to be replicated. But if true, it speaks to how difficult conversations about the topic with next of kin can be and how allowing space for grieving is critical. On the other hand, from a brain tissue quality perspective, the lower the PMI, the less degradation will have occurred and therefore the more valuable the tissue will be for research purposes. Several of the studies had anecdotes from next of kin noting disappointment regarding the conversation they had about brain donation. This difficult conversation also needs to occur at the right health literacy of the next of kin and address any concerns they may have, such as the impact of donation on funeral practices.

Three challenges in interpreting neurogenesis data from banked human brains

One field where the methods of studying postmortem human brain tissue have been relevant recently is adult neurogenesis.

In 2018, Sorrells et al made a splash when they used brain samples from 37 donated brain samples and 22 neurosurgical specimens from people with epilepsy to suggest that neurogenesis only occurs at negligible levels during adulthood. This data seemed to contradict results from rodents.

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DCX staining in rats; Oomen et al 2009 10.1371/journal.pone.0003675

I recently came across Lucassen et al 2018, which critiques Sorrells et al 2018 on a few methodological grounds:

  1. Postmortem interval: Very little clinical data was made available for each brain donor in Sorrells et al, and the postmortem interval (PMI) was one of the omitted variables. The neurogenesis marker DCX appears to be broken down or otherwise be negative on staining shortly after death, so these extended PMIs could cause false negative for DCX staining. Lucassen et al also noted that there might be differential effects of PMI in old and young human brains, for example as a result of differences in myelination.
  2. Cause of death: Lucassen et al noted that certain causes of death, such as sepsis, might be more likely to cause a breakdown of protein post-translational modifications. In the case of the other neurogenesis marker studied, PSA-NCAM, its poly-sialic group might have been lost in hypoxic brains that have substantial perimortem lactic acid production and resulting acidity.
  3. Need for 3d data: Lucassen et al note that the individual EM images presented by Sorrells et al are difficult to interpret because brain cells have complicated, branching morphologies. Instead, they suggest that 3d reconstructions of serial EM images would be more dispositive. Creating 3d reconstructions is often more difficult to accomplish in postmortem human brain tissue compared to rodent brain tissue if the cell processes span a volume that is too large to be effectively preserved by immersion fixation and perfusion fixation is not possible.

I don’t know enough about human neurogenesis, DCX, PSA-NCAM, or the other areas discussed to know if Lucassen’s critiques mean that Sorrells et al’s data truly won’t replicate. But I found the methodological critiques to be valid and important.

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):

Screen Shot 2019-06-25 at 9.26.00 PM
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.

Screen Shot 2019-06-25 at 9.40.06 PM

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.

Screen Shot 2019-06-25 at 9.57.16 PM

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.