Notes on delivering preservative chemicals into the brain

• Link to the index: https://brainpreservation.github.io/

Summary

With one exception (unprotected cryopreservation), all of the preservation methods I will discuss in these essays rely on delivering preservative chemicals into the brain. The principles are similar for cryoprotectants, fixatives, embedding agents, or other chemicals, with some differences due to chemical properties such as diffusion speeds and viscosity.

Time is of the essence. The longer it takes for preservative chemicals to reach their targets, including areas deep inside the brain, the more brain structure will have decomposed, potentially leading to information loss.

Most of the available preservation techniques result in good structural preservation quality in small biological tissue samples. The major problems are in scaling those techniques from very small samples to the size of the whole human brain.

One common method to try to get preservation agents into the whole brain is perfusion. Perfusion refers to cannulating part of the vasculature system and then applying pressure to drive preservative-containing fluid through the blood vessels, from which it travels out into the tissue. Perfusion can also be performed through the ventricular system in the brain.

The other major method is immersion. Immersion refers to placing the brain or body in a large bath of preservation agent, which then travels into the tissue from the outside in. There are a variety of techniques to speed up immersion preservation, such as injecting the preservation agents into the central areas using a needle.

Stabilization

Stabilization is the term used in cryonics for procedures used for the time between when (a) someone is declared legally dead and (b) a definitive preservation procedure, such as subzero cooldown, cryoprotectant agent perfusion, or fixation is initiated. These methods sometimes include include cooling the brain, cardiopulmonary support (which is similar to CPR, but with the goal of providing oxygenated blood to the brain rather than revival), the infusion of anti-clotting agents, and, in cases with access to the necessary technology, the use of extracorporeal membrane oxygenation (ECMO) (de Wolf et al., 2019).

In my opinion, cooling the head and brain after legal death to refrigerator temperatures is currently the most important and “high-yield” intervention in stabilization. Cooling is routinely done in brain banking, pathology, and funeral homes in order to decrease the rate of tissue degradation. This can be accomplished by placing ice around the head and/or by placing the body in a refrigerated room.

It is useful to qualify the amount of postmortem time that has occurred by specifying the extent to which the brain was cooled during this time period, i.e. warm ischemic time vs cold ischemic time, because the former is so much more damaging.

Methods to speed up the actual brain cooling, which is quite slow, would be valuable, although this is a difficult problem to solve. One study found that ice doesn’t tend to form quickly in brain tissue until the temperature is -9xC to -14xC, suggesting that storing at a lower temperature than 0xC by a few degrees for an initial window of time during the initial stabilization period might be helpful. This could increase the cooling rate without causing the damage effects of ice (Zhang et al., 2018). This requires further research, however.

Regarding other stabilization methods, one study found that pentobarbital treatment led to a two-fold increase in neuronal survival in culture 2 and 4 hours after death (Viel et al., 2004). However, this was found to be less effective than ice treatment.

Stabilization methods would require a book or long essay discussion of their own. They will not be discussed here further. For now, we continue on to preservation methods. For more on this topic, see: (de Wolf et al., 2019).

Background on perfusion

Preservation methods are already quite effective and relatively easy to perform in small organisms or small organs. It is clear that it is possible to preserve small sections of brain tissue with minimal damage, using either fixation or cryopreservation, such that the vast majority of the biomolecule-annotated connectome is likely retained. If only the brain was the size of the nematode C. elegans, long-term suspended animation of the brain would already be possible, these essays could be a lot shorter, and I could spend my free time relaxing on a beach instead.

However, scaling up these preservation methods to large organs such as the human brain has proven difficult. A key reason for this is that it’s hard to consistently get the amount of preservative chemicals needed into the tissue quickly enough, before the tissue suffers decomposition damage.

Resource-unlimited, ideal-scenario methods for solving the scaling issue in brain preservation are almost all based on the pressure-driven distribution of preservative fluids through the brain’s vascular system. This is known as perfusion. The major alternative to perfusion is immersion preservation. Immersion generally refers to methods which allow the preservative chemical to diffuse into the brain tissue without accessing the blood vessels.

The idea behind perfusion is simple: during life, the bloodstream supplies oxygen and nutrients to all areas of the brain by an extensive vascular system. At least theoretically, the vascular system can be used during brain preservation as well to distribute preservative chemicals to the brain.

The brain is a relatively well vascularized organ, accounting for around 15% of the body’s cardiac output but only 2% of the body’s volume (Xing et al., 2017). There is an average distance between microvessels of just 20 um in the gray matter and 30 um in the white matter (Schlageter et al., 1999). Almost every neuron has been reported to have its own capillary (Spencer et al., 2007). So based on circulatory density alone, the possibility of perfusing the brain for preservation is appealing.

One researcher told me that a useful way to think of perfusion is like immersion but where you’re peeling open the entire vascular system to increase the surface area by 100- or 1000-fold.

Perfusion-based preservation of the brain immediately at the time of legal death has been reported to lead to “extraordinarily good results” (Trump et al., 1975). We are lucky that the brain perfuses so well, because this is not necessarily the case for all organs. For example, part of the microstructure of the kidney – the lumens of the proximal tubules – has been reported to be lost within seconds of the interruption of circulation. As a result, even immediate perfusion-based preservation does not yield good results (Trump et al., 1975).

Achieving high-quality perfusion is helpful for nearly every method of brain preservation. Some have argued that the quality of perfusion is more important than choosing between different methods, such as choosing between pure cryoprotective agent perfusion versus aldehyde-based methods.

History of perfusion-based preservation

Perfusion is certainly not new. Injection of preservative chemicals into arteries was practiced by several anatomists in the Renaissance period (Brenner, 2014). Frederik Ruysch (1638-1731) was one of the first to get good success with perfusion via injection, although they kept the details of their method a secret (Brenner, 2014).

A book by Thomas Pole (1753-1829) on anatomy notes that trans-thoracic cardiac perfusion was used to perfuse the whole body (Pole et al., 1790). However, this was discouraged for adult humans because they were too large which made the perfusion more cumbersome and less effective. The preferred age range for whole body perfusion preservation were the bodies of people who were 14 years or younger at the time of death. Pole also described bilateral intra-carotid perfusion following isolation of the head at the sixth or seventh vertebrae.

With the advent of formaldehyde fixation as a preservation method in the 1890s, there was a more powerful chemical available to use for perfusion-based preservation. In the early 1900s, the Sicilian embalmer Alredo Salafia (1869–1933) was among the first to perfuse formaldehyde as a preservative chemical, alongside glycerin, alcohol, zinc chloride, and zinc sulfate. One of the bodies that Salafia preserved, the body of Rosalia Lombardo, who was two years old at the time of her death in 1920, is one of the best preserved bodies of that era. A CT scan performed in 2010 showed that Lombardo’s macroscopic brain structures were still intact, albeit significantly shrunken (Panzer et al., 2013).

By 1936 the relative benefits of perfusion-based preservation of the brain in laboratory animals had been reportedly so well established that (Bodian, 1936) wrote:

In spite of the fact that it has been demonstrated time and again that fixation by perfusion with fixing fluids is generally superior to immersion fixation, some investigators continue to state that the contrary is true. It might be well therefore to stress again the importance of adequate perfusion as a method of fixation, particularly for nervous tissues. Failure to achieve better results with perfusion than with immersion fixation is must often due to improper execution of the technique of perfusion.

In 1954, the eminent pathologist Ralph Lillie (Lillie, 1954) wrote that “from the point of view of the histologist, the practice of hardening an entire human brain without perfusion, by immersion in dilute formaldehyde solution or other fixative before dissection, can only be condemned.”

In the 1950s and 1960s, neuroscientists began to use electron microscopy to study the “fine structure” of the brain. In a series of papers in the mid-1960s, Ulf Karlsson and Robert Schultz helped to definitively establish the use of perfusion-based preservation with formaldehyde and glutaraldehyde as the gold standard method for fine structure brain preservation (Ulf Karlsson et al., 1964) (Robert L. Schultz et al., 1965) (as cited in (Korogod et al., 2015)). They described the deficiencies of immersion-based methods in only preserving fine structure well at the surface of the brain and how perfusion helped to distribute fixatives throughout the whole brain.

Neurocirculatory anatomy

From the heart, the ascending aorta eventually branches into the arteries that supply blood circulation to the brain: the carotid arteries and the vertebral arteries (Kelley et al., 2022):

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The left and right internal carotid arteries branch into the anterior and middle cerebral arteries and bring blood to the anterior and middle parts of the brain:

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The left and right vertebral arteries supply blood to the posterior parts of the brain. They travel in the more posterior part of the neck, ascending in the transverse foramen of the cervical vertebra:

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The carotid and vertebral blood vessels supplying the brain meet and anastamose in an area in the center and inferior (bottom part) of the brain, known as the circle of Willis:

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Theoretically, the collateral circulation in the circle of Willis is an opportunity for fluid entering the brain through the carotid system to enter into the vertebral system, and vice versa.

In practice, the amount of collateral circulation is usually limited. Some people don’t have good circle of Willis collateral circulation even during life. One study found that more than 90% of brains from elderly people had at least one hypoplastic component and more than 90% had atherosclerosis (Wijesinghe et al., 2020). Circle of Willis vessels decompose during the agonal and postmortem periods, just like other blood vessels in the brain.

There are some diagrams showing what parts of the brain are generally thought to be supplied by the different blood vessels. For example, here is a diagram of the supplied areas at the level of the superior lateral ventricles:

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As you can see, the carotid artery system supplies the majority of the anterior and middle parts of the brain, while the vertebral artery system supplies the major of the posterior part of the brain. In practice, the areas supplied are more variable and more overlapping than these images suggest.

One factor to consider is that the carotid arteries are much larger caliber and they are found more anteriorly in the neck compared to the vertebrals. As a result, it’s a lot easier to access and cannulate them for perfusion via neck dissection.

Perfusion fixation of human brains has also been reported via perfusion through the femoral artery, followed by “post-fixation” via immersion (Alkemade et al., 2020):

Tissue was routinely fixed within approximately 24 h after death using perfusion fixation through the femoral artery with a 2.3% formaldehyde concentration with 20% ethanol (V/V) for perfusion, followed by 2.3% formaldehyde concentration with 10% ethanol (V/V) for post fixation for 30 days. A small amount of methanol is added to prevent spontaneous condensation polymerization. Ethanol is used for fixation and permeabilization of fatty substances without solubilizing them, 8.3% glycerol (V/V) is added to reduce stiffness of muscle tissue. Tissue fixation is therefore based on an initial alcohol fixation, which is followed by a cross-linking reaction (Fox et al., 1985).

Different brain perfusion methods used

There are a number of different perfusion methods used in human brain banking. A team of investigators that I was a part of reviewed the different methods that have been used for perfusion fixation in human brain banking, resulting in our 2019 review article.

These are the major classes of methods that have been used for blood vessel access, and brief summary of the upsides/downsides of each:

	Description	Upsides	Downsides
Ex situ	Brain removed from the skull	Easier to monitor perfusion quality + Easier to use immersion methods at the same time	Damage to brain and blood vessels when removing the brain from the skull + Introduction of fat emboli (J. Cammermeyer, 1977)
In situ (common properties to all types)	Brain inside of the skull	Minimizes trauma due to brain removal prior to preservation	Difficulty perfusing the brain in the context of brain edema + More difficult to monitor preservation quality
In situ, head separated	Head separated from the rest of the body	Allows faster cooling	Introduction of air bubbles (Shatil et al., 2016) + Disgust reaction
In situ, neck dissection	Vessels cannulated within the neck	If cannulating only the carotids, likely the fastest way to begin perfusion	Difficulty in cannulating neck vessels, especially the vertebral arteries
In situ, thoracic dissection	Aorta cannulated in the thorax	Allows one cannulation to perfuse all four cranial arteries	Difficulty in ensuring that chemicals reach the brain as opposed to the rest of the body

Monitoring preservation quality is a problem for the in situ approaches. One option is to test the tensile strength of the eyes, which will harden as fixation of the eyes proceeds. Another approach is to monitor the effluent fluid that returns to the neck via the jugular veins, such as via measuring its refractive index.

A more effective way to monitor preservation quality in the in situ approaches is likely to open up at least part of the skull, for example via a burr hole. Or to use non-invasive neuroimaging.

Alcor’s method for brain-focused cryopreservation is to separate the head from the rest of the body in a cephalic enclosure and cannulate both of the carotid arteries for cryoprotectant agent perfusion (de Wolf et al., 2019). If there is evidence that the circle of Willis is damaged, then the vertebrals are cannulated and perfused as well. In addition to having benefits for perfusion monitoring, separating the cephalon also allows for more rapid cooling rates.

The Cryonics Institute’s standard method for brain-focused cryopreservation is to cannulate all four of the carotid and vertebral arteries through neck dissections with the whole body intact. The perfusion quality in the brain is monitored through burr holes in the brain. Historically, they have found that in some cases, attempting to perfuse the rest of the body as well with cryoprotectants seemed to cause fluid to enter the brain from the rest of the body, with potential damage due to the different osmotic concentration of that fluid (from here):

After CI’s 77th patient it was decided to never perfuse the body, but this proved to be too unpopular with CI Members. Perfusion of the body of the 77th patient with 80% ethylene glycol had achieved little other than loading the abdomen with cryoprotectant. Worse, the core temperature of the brain rose more than the surface temperature of the head in the 77th patient – indicating flow of fluid from the body into the head and brain. The potentially destructive effect of body fluids entering a perfused brain osmotically (which caused us to elevate the head of the operating table for this patient) is all the more a threat as a result of the pressure generated in perfusing the body with cryoprotectant. Glycerol perfusion of the body seems to be safer for the brain, and CI Members now can choose to have body perfusion, although head-only perfusion is the default.

Problems with perfusion

Perfusion is an obvious choice for brain preservation. It’s the gold standard method in laboratory animal studies for preserving the fine structure of the brain. And perfusion is clearly the most promising approach in the nascent field of organ cryopreservation. So why isn’t this a slam dunk for use in brain preservation? Unfortunately, there are a few problems with perfusion that can limit its effectiveness in practice.

Humans are not as healthy as laboratory animals at legal death

Humans who have experienced legal death are much different from laboratory animals.

First, they tend to be much older. With age, capillary density decreases, which is likely to make perfusion less effective. “A man is as old as his arteries,” as Sydenham said.

Second, partially related, the prevalence of cerebrovascular disease at legal death is extremely high. One or both carotid arteries may have significant stenosis. The microvessels within the brain are also likely to be substantially less patent. Cerebrovascular disease will limit the movement of preservative chemicals, make it more variable across the brain, and also harder to predict.

Third, the cause of legal death in humans almost always cannot be controlled and may present a problem. For example, if people legally die of a stroke, especially a hemorrhagic stroke, then parts of their cerebrovascular system may be completely compromised even prior to legal death. Clearly those vessels cannot be employed for preservation by perfusion. Other causes of legal death can lead to significant formation of thrombi, various forms of emboli, vasoconstriction, or other factors that will hinder adequate perfusion after legal death. For more, see the essay about the agonal phase.

Even when the cause of death can theoretically be controlled, as it is for people who live in jurisdictions that allow Medical Aid in Dying via prescribed medications, it can take a long time for legal death to actually occur. The time it takes is on the scale of hours, including one case that lasted 104 hours. During that time, the brain will likely be undergoing hypoxia and agonal decomposition. This is a clear contrast to the many animal studies where the perfusion of fixatives is the cause of death in anesthetized animals.

As a result of these and other factors, studies that describe a preservation procedure in animals in controlled laboratory settings cannot necessarily be extrapolated to practical human cases. This is a critical point that we need to keep in mind while we evaluate this literature.

Postmortem decomposition significantly hinders perfusion

There are many sources of postmortem delay prior to brain preservation, the main two being:

1. Dying far away from people who can perform the preservation procedure.

A sudden unexpected cause of death occurs in around 10% of deaths (Mary Elizabeth Lewis et al., 2016). A sudden death can substantially increase the postmortem interval because people tend to be far away from a team of people who can begin the preservation procedure. People also frequently legally die while others think they are asleep, before anyone notices. For people who live alone, it can be days before someone notices that they have legally died.

Even in the case of expected legal death, people who are interested in brain preservation are so few that most people do not die in a location where they can access this procedure soon after their legal death.

2. Medicolegal delays. While people are alive, they often lack body autonomy as a result of societal medicolegal frameworks. After legal death, in all jurisdictions that I am aware of, people can have very limited ability to decide what happens to their bodies. If the legal death is declared due to something other than “Natural Causes”, the state effectively has total control of the body until it is released, with no way of opting out of this process. This can result in involuntary autopsy and/or medicolegal delay that could on their own be responsible for information-theoretic death. This will be discussed much more later in these essays.

Blood vessels are just like any other part of the brain when it comes to decomposition. As soon as they stop being oxygenated, the cells and extracellular structures that make up the blood vessels start to decompose. Eventually, they turn into a liquid-like state. So with enough postmortem decomposition, perfusion is going to be useless. Or even harmful to attempt it.

The amount of decomposition that can occur before perfusion-based preservation is no longer a good idea is a very poorly understood problem. Here are a few data points:

• Bodian noted that perfusion needed to be started within 5 minutes after the death of the animal (Bodian, 1936).
• One analysis in cryonics suggested that keeping warm ischemic time to less than 8 minutes was predictive of less cerebral edema during perfusion-based preservation [as cited in (Darwin, 2011).
• While there is little formal data in perfusion fixation in human brain banking on this topic, some investigators have personal experience and opinions. One investigator in brain banking I spoke to suggested that perfusion fixation more than 6 hours after legal death was not worthwhile. But another told me that it was still worthwhile to perform up to 24 hours after legal death.
• Karlsson 1966 compared perfusion fixation at immediate death to 5-60 minutes afterwards and reported that in the delayed cases, the perfusion rate was slower, the brain tissue was sometimes softer when dissected out, and the typical color was not always found (Ulf Karlsson et al., 1966):

“Generally, delayed perfusions of exsanguinated rats (”dead” animals) flowed at a slower rate than perfusions of immediately living animals. Also, the initial muscular reaction was weakened or absent. Some brains of the investigated experimental material appeared softer than the normal material (13) when dissected out. A concomitant pallor was sometimes observed instead of the usual yellowish tint of fixed, glutaraldehyde-perfused brain tissue. This softness and pallor were inconsistently found.”

• William Hunter (1718 - 1783), who was one of the first to do embalming via vessel injection, understood that it was critical to begin the perfusion process early. He also recognized that if the tissue was cooled, it could wait longer. He advised that the embalming process should begin within 8 hours of death during the summer and within 24 hours of death in the winter. He called this “his most critical advice.” (Bryant, 2003)

• Cammermeyer 1960 noted (J. Cammermeyer, 1960):

“Admittedly, fixation by perfusion is difficult and a slight error in the procedure, in particular with small animals, may result in conspicuously poor preservation (5) and severe cell changes which may be interpreted erroneously as pathological. Fortunately, there is sufficient contrast between well and poorly fixed parts when Heidenhain’s Susa or Bouin’s picric acid solutions are used, permitting the selection of only well-fixed tissues for cytological studies. The latter should be essential for any kind of morphological study, whether light or electron microscopes are being used (16). It may not always be practical or feasible to utilize perfusion. In fact, the immersion procedure is preferable when the object of a study is to determine the distribution of emboli, thrombi, and hemorrhages (22). The technique of choice is handicapped in clinical material because of pathological vascular changes causing undue permeability to perfusates and because of the delayed autopsy with resultant autolysis and increased hydrophilia. In such materials the abnormal reaction of the tissues to isotonic saline (54, 1) will provoke the perivascular accumulation of water, pseudoedema, which by the compression of blood vessels will compromise further flow of the fixative (8, 9, 32).

One of the major reasons for poor perfusion quality following a delay in the start of the procedure after legal death is the “no-reflow” phenomenon. This refers to the finding that after blood flow to the brain is stopped, it is often not possible to restart it.

• Sheleg 2008 describe the no-reflow phenomenon as such (Sheleg et al., 2008):

Why is it almost impossible to resuscitate a patient with prolonged warm cardiac arrest? There are two possible explanations. The first one is the “no-reflow” phenomenon. The “no-reflow” phenomenon was described by Ames et al in 1968 [16]. Brain ischemia causes some degree of edema, which closes the microcirculation due to the pressure on the capillary wall from outside. In the reperfusion phase, the larger branches are perfused again, but the microcirculation remains closed due to the persistent edema.

It is also worthwhile to point out that the no-reflow phenomenon has also been criticized in the literature as an explanation of all neuropathologic damage following cerebral ischemia (Brierley et al., 1973).

Certain brain regions are more difficult to perfuse

In general, white matter has been found to be more difficult to perfuse than grey matter. For example, in 1992, Mike Darwin reported that when Fahy attempted to vitrify dog brains, he was only successful in vitrifying the gray matter, not the white matter:

[W]hen Fahy attempted to vitrify dog brains using his DMSO-propylene glycol(PG)-formamide mixture he was successful in vitrifying only the gray matter. The white matter was extensively (perhaps uniformly) frozen. Why? Because the myelinated tracts are not well penetrated by cryoprotectant. Perhaps they are only not well penetrated by the formamide and PG. Additional research needs to be done to answer this question.

In part, this could be because white matter is less well vascularized than grey matter. Or in part, this could be because it’s more difficult for the preservative chemicals to penetrate myelin, which is a major component of white matter.

Another consideration is the presence of the blood brain barrier. The relative difficulty of perfusing chemicals across the blood brain barrier was actually how the blood brain barrier was first discovered. It was found that injecting dyes into the circulatory system stained the rest of the body, but not the brain, and that this could be circumvented by directly injecting dye into the brain tissue (Saunders et al., 2014).

Kalimo 1976 reported that regions without a blood brain barrier but instead with fenestrated capillaries, namely the median eminence of the hypothalamus, the anterior pituitary gland, and the posterior pituitary gland, have different outcomes during perfusion fixation (Kalimo, 1976). Specifically, perfusion with an isotonic osmotic concentration led to shrinkage of cells and the extracellular space in these regions, while it led to good preservation of the regions that do have a blood brain barrier. They reported that “the distinct boundary zone between the well-preserved hypothalamus and the badly destroyed median eminence clearly demonstrates the different behaviour of the BBB and endocrine regions during the fixation.” Because poor preservation in the regions with fenestrated capillaries could be prevented by using a preservative buffer with a different osmotic concentration, the poor preservation was attributed to osmotic damage in those regions. I haven’t seen another study report this finding and the finding is a bit “cute”, so it should be taken with a grain of salt.

Edema and osmotic forces

Osmotic damage is a major potential problem in perfusion-based approaches. Let’s review what osmosis is. Osmosis is the movement of solvent molecules (usually water) across a selectively permeable membrane (such as a cell membrane) to a region of higher to lower osmotic concentration:

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Rapid movement of water across cell membranes or other tissue structures as a result of osmotic concentration differences can cause significant damage to the tissue. Osmotic damage is a feared outcome in certain clinical contexts involving rapid shifts in the circulatory osmotic concentration, for example leading to osmotic demyelination syndrome (King et al., 2010).

Because the preservative chemical is rapidly introduced into the brain by perfusion, any osmotic concentration difference between the preservative chemical and the brain tissue will manifest rapidly, as opposed to more slowly as would occur in immersion-based approaches.

It is hard to estimate what the osmotic concentration is in the brain. It is different between individuals, it differs across the postmortem interval, and it is different across brain regions. So it is hard to identify what the osmotic concentration of the preservative chemical should be, although there may be an acceptable range that will not cause significant damage.

Our review noted that hypertonic fixative solutions are liable to cause cell shrinkage, while hypotonic solutions can cause edema (McFadden et al., 2019). One of the reasons that edema is a problem is that it can compress the brain tissue against the closed space inside of the skull, causing mechanical damage. As our 2019 review noted:

One potential problem with the use of a washout solution in brain perfusion fixation is that it may induce brain edema. In animal studies it has been shown that perfusing too much saline into the brain (e.g., one liter) can cause edema [11]. The edema induced may be related to the osmotic concentration of the washout solution. Consistent with this, Benet et al. [9] found that washing out with an isotonic saline solution rather than tap water led to decreased tissue edema. Grinberg et al. [34] compared a hyperosmolar solution of 20% mannitol with a solution of 0.9% NaCl, finding that 20% mannitol led to substantially less brain swelling.

Another problem with edema is that it can cause cells to change their morphology and take on an artifactual appearance, sometimes known as “hydropic cell change”. This is described in the neuropathology literature as occurring if a saline washout step in perfusion fixation is too protracted (Brierley et al., 1973):

“Hydropic cell change” or “water change” is an artifact studied in detail by Jakob and by Cammermeyer. It is sometimes seen in the human brain and particularly in infants. It occurs in the immersion-fixed animal brain but is a particular hazard of perfusion fixation if the preliminary saline washout is protracted. Typically, the hydropic cell is swollen and the cytoplasm stains less intensively than normal. This is most marked in the periphery of the soma which has a fuzzy outline and may contain one or more large vacuoles. The corresponding electron microscope appearances include swelling of the membranes of the endoplasmic reticulum, ballooning of peripheral mitochondria, and rarefaction of the granular endoplasmic reticulum.

The authors of one study argue that, at least when perfusing fixative immediately after a rapid death in mice, there is no advantage of using a washout prior to perfusing fixative (Tao-Cheng et al., 2007):

Our results with fast perfusion fixation demonstrate that, in mice, there is no need to flush out the blood to prevent blood clotting prior to fixative perfusion and that perfusion directly with fixative may prevent dynamic structural changes that might occur during the period of flushing.

Osmotic concentration differences also have the potential to cause significant damage. There is evidence that fixatives do not introduce such a strong osmotic pressure, whereas cryoprotective agents do. As a result, perfusion with cryoprotective agents is likely more concerning regarding the possibility of osmotic damage.

Grinberg 2008 also note swelling after perfusion. For this reason, they preferred to use mannitol for the washout (Grinberg et al., 2008):

For preventing hardening of the blood in the vessels, blood must be washed out from the vascular system before perfusion fixation. However, when the blood was washed out using NaCl 0.9%, the brain showed a irreversible swelling, especially after formalin perfusion (Table 3). We do not have a clear explanation for this phenomenon; however, previous authors report identical results after whole brain perfusion. Kato found an average of 20% increase of weight in brains perfused with 10% unbuffered formalin. Even after 5 months of fixation the average weight was 14% greater than the original (1939). Frontera reports similar results (1959). Perhaps it can be explained either because isotonic saline induces the movement of extracellular sodium, and chloride ions and water into neurons provoking swelling of the latter (Van Harreveld and Steiner 1970), or by the rapid penetration of formalin in the form of methylene glycol into the tissue (Fox et al. 1985). Other studies report less brain swelling after formalin perfusion. The reason may be the use of buffered formalin (Beach et al. 1987) or the mediosagittal severing of the brain prior to fixation (Waldvogel et al. 2006). Our staining and 3D reconstruction protocols require unbuffered fixative and whole brain fixation, respectively. Therefore we did not test these alternatives. An important point is that the fixated specimen can be re-scanned ex-situ and then we can provide better information regarding the fixation effects in the MR signal and volume – at the same time, this would provide a better matching with the histological images.

It has been suggested to replace saline by a substance that is unable to enter the cells in order to prevent swelling of the latter (Cragg 1980). Mannitol, a sorbitol stereoisomer, works as an osmotic diuretic. Its osmolarity and its capacity to open the blood-brain barrier by transitorily shrinking the endothelial cells make mannitol a better substance for washing the blood out. Mannitol-formalin perfusion induced less swelling, but still 15% over the initial weight. AAF perfusion fixation preceded by mannitol rinsing gave the best results with an average swelling of 6% in our cases (Table 3). Using this protocol, brain deformation was minimal and tissue preservation was excellent.

One study suggests that using a hypertonic perfusate solution may be helpful to counteract the high hydrostatic pressure associated with perfusion and thus prevent edema (Høyer et al., 1991):

Non-fixing compounds may be added to the fixative to contral osmolarity (salts, sucrose), stabilize membranes and reduce the extraction of lipids (Ca2+), or to give the vehicle a high colloid osmotic pressure (dextran, polyvinyl pyrrolidone). This last approach appears to limit changes in volume that tend to occur between endothelium and epithelium with perfusion fixation in loosely built tissues such as kidney, panereas, and intestine (Bohman and Maunsbaeh, 1970). The high colloid osmotic pressure probably counteracts the high hydrostatic pressure caused by the perfusion (Fig. 12.4). This is analogous to the normal transport of material across the capillary wall (Fig. 12.5): About half way through a capillary the hydrostatic pressure is equal to the colloid osmotic pressure. In fixing tissue by vascular perfusion without the addition of colloid, the hydrostatic pressure is, in principle, sustained without the counterbalancing influence of colloid osmotic pressure. Fixation, therefore, tends to produce an increased extracellular space (Fig. 12.4) just as oedema occurs in conditions where blood protein levels are reduced (e.g. proteinuria).

Perfusion is technically challenging

Perhaps the biggest problem with perfusion is that it’s difficult. It requires trained experts and relatively expensive machinery.

As an example of the technical challenge, let’s consider the in situ, neck dissection approach. Ideally, cannulating the blood vessels of the neck means cannulating four blood vessels: the carotid and vertebral arteries on each side of the neck. Accessing the vertebral arteries in particular is time-consuming and technically challenging.

Alternatively, one could not cannulate the vertebral arteries, which would save time and requires less expertise as they are much more difficult to access than the carotids. But you then either: (a) rely on an adequate anastomotic circulation between the anterior and posterior circulatory system of the brain in the circle of Willis or (b) accept that the posterior regions of the brain, like the cerebellum and visual cortex, will not be as well preserved by perfusion.

An alternative approach is to isolate the cephalon from the rest of the body and then cannulate the neck vessels. This also takes time, it’s still not very easy to cannulate the vessels, and it could theoretically introduce air bubbles into the circulatory system, which could decrease perfusion quality. And it might also horrify some people, because of the innate human reaction to separated cephalons. But it is likely easier than neck dissection to access all four blood vessels. One study found that separating the head prior to perfusion seemed to be helpful for preserving the elephant brain (Manger et al., 2009).

Notably, Cammermeyer 1960 found that exposing the brain to air prior to perfusion did not affect perfusion quality, provided that the delay was not too long (J. Cammermeyer, 1960).

Perfect perfusion and fixation were obtained, even if the brain was exposed to air and thus to atmospheric pressure prior to death or if perfusion was performed 10 min after death. “Dark” neurons were present in areas of imperfect perfusion. They were present when the perfusion was delayed 30 min. For proper fixation many factors had to be considered; among them, hydrostatic pressure, composition of the solution to flush blood vessels, use of a coagulant fixative, and delayed autopsy. The aim of fixation is to affix cellular membranes in such a manner that neurons are no longer vulnerable to traumatization incurred during autopsy and removal of the organ.

Additionally, neck vessels don’t just supply blood to the brain. They also supply the rest of the cephalon, such as the face. This means that even if you perfuse preservation chemicals through the carotid arteries in the neck, you might think that you are perfusing the brain via the internal carotids, when in reality you are mostly perfusing blood vessels in the face via the external carotids. Which, obviously, is not the focus. In order to prevent this, some investigators have advocated to clamp the external carotid arteries to prevent preservative fluid from flowing in this direction. But that adds additional complexity and time and might not be effective.

Another approach is to cannulate the thoracic aortic artery. While this is easier to perform in a smaller animal such as a mouse or a rat in a laboratory setting, it has been frequently reported that it is difficult to perform in a large animals, such as pigs (Musigazi et al., 2018), elephants (Manger et al., 2009), or humans.

As a sign of how challenging perfusion is, consider that even in controlled animal experiments with trained personnel starting the procedure immediately at the time of death, sometimes perfusion does not work, for apparently idiosyncratic reasons. For example, here is part of the detailed methods section from (Tao-Cheng et al., 2007):

Brains for immuno-EM were dissected and examined as soon as perfusion fixation was finished. If the brains were soft, or pink they were discarded. Only hardened brains cleared of blood were accepted and further fixed by immersion in 2% paraformaldehyde for a total of 60 minutes of fixation (counted from the start of perfusion fixation).

Discarding the brain if the perfusion does not work is not an option in brain preservation. Every preservation is important.

The technical challenges in perfusion-based methods limits access to brain preservation. Some people think that perfusion is necessary to achieve high enough brain preservation quality for eventual revival. Personally, I’m not as sure, because I think there is considerable uncertainty about what the required level of preservation quality is, as discussed in the essays in the “Metrics” section.

Perfusion quality metrics

How can we evaluated the quality of perfusion? Here are some metrics that some studies have used.

(Frigon et al., 2022):

• Brain color
• Dilated vessels on microscopy
• Neuropil quality (fissured vs not)
• Cell shape (shrunken or not)
• Antigenicity preservation

(Wohlsein et al., 2013)

After successful perfusion, CNS vessels lack blood cells, and the perivascular space is no longer visible. Similarly, both aforementioned parameters are useful in evaluating whether perfusion was performed properly.

(Jenkins et al., 1979)

All perfused brains utilized in this study were of uniform color and firmness and displayed no macroscopic evidence of red blood cells in the brain vasculature.

(Garcia et al., 1978):

The adequacy of the fixative flow, following MCA occlusion, was evaluated as follows: a, in some animals, pial reperfusion was recorded photographically (Sundt et al., 1969), b, in all animals reported herein the electron-dense tracer added to the terminal portein of the fixative (Garcia et al., 1977), was visible in the lumen of all capillaries in all areas studied, and c, biochemical parameters reflecting the adequacy of the flow were developed after creating situations of either global or regional ischemia The results obtained after MCA occlusion (7 h duration or less) are indicative of variable degrees of perfusion abnormalities, by both blood and fixative, but not of an absence of fixative penetration.

(Monroy-Gómez et al., 2020)

Macroscopic differences were observed between the brains of the mice fixed by perfusion and those fixed by immersion. The perfusion extracted completely the blood in the brains, and gave them a hard consistency facilitating their extraction from the skull and obtaining the slices. In the brains of the animals sacrificed by euthanasia and immersion-fixed, large amounts of blood in the tissue and a soft consistency was observed, the latter increased with the postmortem degradation time. This complicated the extraction and the execution of the cuts in the vibratome.

(Krinke et al., 1995): A nice abstract on perfusion quality metrics, which seems to track the extent of autolytic changes in the dorsal fascia dentata, which is reportedly an autolysis-sensitive region.

Another study on perfusion fixation quality metrics in animals basically characterizes poor perfusion as areas with worse postmortem changes (Dehghani et al., 2018).

One study on perfusion quality in animals found that MAP2 visualization was no different in perfusion vs immersion fixed brains (Bay et al., 2021)

Variations on the perfusion methods

Washout chemicals and the use of anticoagulation

One of the commonly used approaches to try to improve perfusion is to use a different set of preservative chemicals to prepare the vessels prior to the definitive preservative chemicals are perfused. This step is called “washout”. As (McFadden et al., 2019) reports for the case of perfusion fixation: “This step aims to remove clots, blood cells, and other intravascular debris to improve flow of fixative, although it comes at the cost of increased procedural complexity and a longer delay prior to fixation.”

One of the key uses of the washout step is to try to remove clots from preventing the flow of perfusate. This can potentially be aided by the use of a anticoagulant such as heparin, although I have not been able to find any studies with evidence that perfusing heparin or another anticoagulant is actually helpful.

There is some evidence in the liver transplant literature that the use of postmortem thrombolytics in such as tissue plasminogen activator in deceased liver donors can help to decrease the burden of blood clots (Seal et al., 2015). Although trying to apply that data directly to the much different case of the postmortem brain clearly raises challenges.

A blood thinner such as aspirin or heparin might have already been given prior to legal death for other reasons, where it would incidentally likely be much more effective in preventing agonal and postmortem clots. However, the use of premortem heparin is highly controversial even in the case of organ donation (Soares et al., 2011). Given our current laws, brain preservation organizations must maintain complete legal separation from making any recommendations about clinical management prior to legal death.

Perfusion pressure

One of the most important variables in performing perfusion is the pressure. A higher perfusion pressure is associated with a higher risk of vessel rupture. For example, an analysis of 1700 mouse brains preserved by perfusion fixation found that a higher perfusion pressure was associated with a higher risk of liquid accumulating outside of blood vessels and migrating outside of the dura mater (Cahill et al., 2012).

On the other hand, lower perfusion pressure is associated with incomplete perfusion, decreased perfusion in the setting of blood clots, and decreased tissue penetration of the preservative chemical (McFadden et al., 2019).

One study on young laboratory mice in controlled conditions, found that pressures higher than physiologic lead to blood brain barrier disruption and blood vessels dilation, while lower pressures lead to vessel collapse and microclot formation (Schwarzmaier et al., 2022):

While a PP below the physiological systolic blood pressure results in the collapse of parenchymal vessels and formation of microvasospasms and microclots, a PP above the physiological systolic blood pressure dilates cerebral vessels, induces microvasospasms and disrupts the BBB. In terms of tissue integrity, our results confirm that higher PPs lead to fewer artifacts such as dark neurons or perivascular courts.

In this study, lower perfusion pressures were also associated with more histologic changes associated with the postmortem interval, such as perivascular non-staining regions.

Alcor’s perfusion pressure is typically around 80-100 mmHg. They report that exceeding this pressure is particularly problematic in the case of people with brain swelling (de Wolf et al., 2019):

Perfusion pressure during cryoprotective perfusion should not exceed 100 mmHg for both neuro and whole body patients, measured in the arterial line. Because cryoprotectant concentration and lower temperatures both increase viscosity the pump speed needs to be reduced a number of times in the course of perfusion. Not exceeding 100 mmHg is particularly important in ischemic patients and patients with brain swelling. Perfusion pressures below 80 mmHg should be avoided.

Increasing the perfusion pressure may be beneficial in the case of higher postmortem interval, to help overcome perivascular edema and other impediments to perfusion that have accumulated. This requires further research.

Surfactant

In work at the Cryonics Institute from 2001 to 2007, cryobiologist Yuri Pichugin found that adding a small concentration of the detergent sodium dodecyl sulfate (SDS) to the perfusate allowed for the opening of the blood brain barrier. Using SDS has since been found to improve cryopreservation by lowering the amount of dehydration and it has been used by many of the recent perfusion-based brain preservation procedures. For example, in the 2015 paper by McIntyre and Fahy describing aldehyde-stabilized cryopreservation, they stated: “SDS was found to be critical for our purposes by allowing cryoprotectant to penetrate the brain without causing shrinkage. When SDS was included, we found no observable brain shrinkage when measuring brain weight or examining ultrastructure” (McIntyre et al., 2015).

How long of a perfusion is necessary?

(U. Karlsson et al., 1965) found that only 5 minutes was necessary:

As judged by conventional criteria of fixation according to Palade and Sjostrand, it was found that the ultrastructural picture changed very little, if at all, provided that the tissue was perfused by aldehyde for more than 5 minutes. After briefer perfusion, the tissue exhibited all criteria found for a poorly fixed specimen, such as a washed-out appearance, occasional broken membranes, and some plasma membrane separation.

Although this was in rats, which have smaller brains, and with total control of the experiment, so there was no agonal state or postmortem decomposition prior to perfusion. In larger brains and with less ideal perfusion conditions, a longer period of time might be necessary.

Perfusing with low temperature fixatives

(U. Karlsson et al., 1965) found that using low temperature fixative solutions was not helpful and in fact harmful:

No advantage was found in using aldehyde fixative at low temperatures. On the contrary, our results indicated that more reliable results were obtained by using aldehyde perfusates at body temperature for the total perfusion time rather than at first using them at body temperature and later a few degrees above freezing.

One of the potential downsides of low temperature perfusion is that the perfusate will have higher viscosity, and therefore have lower flow rates. It may also contribute to vasoconstriction. Finally, cold fixative will diffuse more slowly once it reaches the tissue. Some have argued that, if anything, the fixative solution should be higher than room temperature. However, I consider this to be an open question. There is no convincing data either way.

Monitoring perfusion quality

The main method used to monitor perfusion quality has been to create a burr hole in the skull and visualize the brain tissue directly. For example, this allows one to monitor whether the blood vessels at the surface of the brain are drained of blood.

One could also imagine using ultrasound to monitor perfusion quality, which would allow real-time monitoring across deeper regions of the brain. This could allow decisions about whether to alter perfusion parameters, such as the perfusion pressure. Theoretically, one could also use neurointerventional radiology approaches to monitor perfusion quality. However, this would require resources far beyond those available in brain preservation today.

Background on immersion

Immersion refers to methods which allow the preservative chemical to diffuse into the brain tissue without accessing the blood vessels.

The main limiting factor in immersion compared to perfusion is the rate of diffusion of the preservative chemical into the brain. This also limits the universe of preservative chemicals that can be used in brain preservation because it requires that they have a reasonably fast rate of diffusion.

To get a sense of scale prior to this discussion, it might be helpful to recall that cells each have a very small amount of fluid in them – approximately 1 picoliter.

Standard immersion method

In the technically simplest form of immersion preservation, the brain is removed from the skull and placed in a container of preservative chemical, which slowly diffuses into the brain tissue and preserves it:

A human brain hemisphere preserved in a solution of formalin; image source

Removal of the brain from the skull takes time, during which decomposition will occur. However, it can be done relatively quickly when people are well practiced in the technique. In one study of pig brains that maintained aspects of brain function, they reported that it took them around 5 minutes to extract the brain from the skull (Vrselja et al., 2019). Chana Phaedra has reported that with practice, removal of the brain could be done within 30 minutes and with little physical trauma. Another study in pig brains reported that it took approximately one hour to remove the brain (Tafrali, 2019).

As expected, removal of the skull has been reported to cause damage to brain if done improperly (Wohlsein et al., 2013):

Forcible removal of the skull may cause distention and artifactual emphysema of the leptomeninx (Eigs. 48, 49) as well as separation of brain structures (eg, molecular layer from Purkinje cell and granule cell layer of the cerebellum), particularly in large animals (Eig. 50).

Removal of the dura is also an essential aspect of immersion-based preservation, for the obvious reason that it removes a key barrier for diffusion to the brain parenchyma.

Penetration speeds in standard immersion fixation

This is a difficult question to address, because it’s unclear how much fixative needs to reach more inner brain regions, and how much time it has to be there, to sufficiently slow the decomposition process. Also, formalin doesn’t have any natural color that distinguishes it from brain tissue. Even if one adds a dye, this will simply penetrate at the diffusion speed of the dye, independently of the formalin. It also depends on various factors, such as the postmortem interval, the temperature (Jones et al., 1992), and possibly the species.

One study using MRI found that fixative penetration occurs at around 1.4-1.5 mm/hr (Nazemorroaya et al., 2022):

Fitting of the Medawar equation (Figure 7G,H) yielded coefficients of 0.93/0.88/1.45/1.51 mm/h1/2 in WM for Fix01/02/03/04, respectively, with adjusted R2 ≥ 0.979 and 0.80/0.77/1.36/1.38 mm/h1/2 with adjusted R2 ≥ 0.912 in GM.

One source notes that formalin penetrates tissue at approximately 1 mm/hour (Hewlett, 2002).

One source notes that methylene glycol – which is the hydrated form of formaldehyde, in chemical equilibrium with formaldehyde in aqueous solutions – may diffuse more faster than formaldehyde (Shiurba et al., 1998).

One source notes that tissue blocks take 24 h for formaldehyde to penetrate at 25°C, or 18 h at 37°C (Thavarajah et al., 2012).

A major upside of immersion preservation is surface preservation

When you look at macroscopic pictures of the brain, the outermost grey matter stands out:

Sections of a human brain showing grey vs white matter distinctions; image source

This outermost grey matter of the brain is a key part of the cerebral cortex. The cerebral cortex is the part of the brain that been the most associated with the higher-order functional aspects of cognition (LeDoux et al., 2020). It has been estimated that there are approximately 17 billion neurons in the human cerebral cortex, many of them in this area (Hart et al., 2008). In most regions of the brain, there are six layers of the cerebral cortex. Sometimes, the cerebral cortex is thought to be horizontally sub-divized into discrete functional units called cortical columns.

In neuroimaging, a functional definition of the cerebral cortex is the area between the pial and white matter surfaces, which is often specifically called the cortical ribbon. The thickness of the cortical ribbon varies across the brain and across individuals, but it has an average thickness of 2.5 mm and tends to be less than 4.5 mm (Fischl et al., 2000).

If formaldehyde pentrates at 1 mm/hour, this means that the surface of the brain will have at least initial exposure to preservative chemicals within 2.5 or so hours of the start of the immersion preservation process. While this is clearly not as fast as the perfusion-based approaches, it is a more robust approach to preservation that does not rely on high-quality perfusion preservation.

Notably, some of the cerebral cortex is contained within folds of the brain. While still a part of the surface of the brain in some sense, these areas are not able to be seen with the naked eye looking at the brain.

Coronal section of thionin-stained human brains demonstrates that some areas of the cerebral cortex are within folds; image source

It seems likely that fluid in immersion-based preservation should be able to penetrate the sulci and preserve the surface areas of those parts of the cortex as well, although not as well, and this is an area of uncertainty.

The ease of preserving the cerebral cortex of the brain with formalin is one of the reasons why immersion preservation is frequently thought of as sufficient for investigations in human brain banking around the world.

One of the investigators I spoke with who uses perfusion-based preservation was interested in inner regions of the brain, such as the thalamus and basal ganglia (a part of which is the lenticular nucleus):

Coronal section of the human brain at the level of the mammillary bodies shows the inner regions of the brain; image source

Both the thalamus and basal ganglia clearly play important roles in cognition. For example, the thalamus seems to be critical in long-term memory recall as it is involved in cortico-thalamic loops.

It has also been previously found that inner areas of the cerebellum are less well fixed by conventional immersion fixation, and this may be exacerbated in the presence of brain edema (Ogata et al., 1986):

Advanced [granular layer autolysis] in the central folia adjacent to the central white medullary body of the cerebellum is assumed to be due to the poor fixation of the central parts of the cerebellum, which is farthest away from the fixative in case of conventional immersion fixation. Especially in the presence of brain edema, [granular layer autolysis] is supposed to develop readily due to poor penetration of the fixative. Because [granular layer autolysis] of the peripheral folia of the cerebellum does not appear in patients who did not exhibit brain death or patients with brain death of short duration, [granular layer autolysis] of the peripheral folia is supposed to develop in the period of brain death.

Some of the rest of the inner areas of the brain is white matter. While clearly essential for cognitive functions, there seem to be more channels of inference to reconstruct the biomolecule-annotated connectome in the white matter, because neuronal processes and myelin can likely be traced and reconstructed with the help of cell compartment-specific biomolecules. So precise preservation may not be quite as essential in white matter – although it’s important to note that the degree of myelination can vary along an axon in ways that are relevant to cognition.

Overall, the surface of the brain seems to contain relatively important information, which is a clear win for immersion preservation approaches. However, there are also some deeper areas of the rest of the brain that also seem critical to preserve. This suggests that there may be a need to perform targeted improvements to the immersion approach that would allow for better preservation of inner areas such as the thalamus.

Problems with the standard immersion method

Removal of the brain causes macroscopic damage

It can be challenging to remove the brain without causing macroscopic damage. This is especially pronounced in the following cases:

• A large degree of postmortem decomposition. In fact, difficulty in removing the brain from the skull without causing damage is one way of grading the amount of postmortem decomposition in Hayman’s classification scheme (Hayman et al., 2017).
• When the brain is warmer. Cooling the brain to refrigerator temperatures causes it to become harder and increases mechanical stability, which is helpful prior to removal.
• Children (or adults less than 20 years old) and people who suffered from cerebrovascular disease in their white matter. This is because their brains have less myelin, and myelin makes brains more firm.

Removal of the brain from the skull can also damage blood vessels, including dural vessels. This can hinder attempts at ex situ pefusion-based preservation because of leakage of the preservative chemicals from these damaged blood vessels.

Removal of the brain from the skull can also cause bone sawdust to accumulate in areas of the brain adjacent to where the skull is accessed:

Bone sawdust in brain tissue; (Rech et al., 2018)

Another problem with removing the brain from the skull prior to preservation is the likely possibility of air emboli in the vessels and air bubbles inside of the brain tissue. Air bubbles will likely mitigate the quality of ex situ perfusion. However, the extent to which air introduction actually decreases perfusion quality is an open question. One investigator who I spoke with that had performed both in situ and ex situ perfusion fixation found that the quality was equivalent between the two.

There are ways to minimize the amount of damage that occurs to the brain when it is removed from the skull. For example, some studies use special containers that the brain is placed into to allow it to retain its shape after removal. Placing the brain directly into a liquid bath would also likely lead to less damage. An initial preservation procedure via perfusion or immersion through a skull window is probably the most definitive way to minimize damage to the brain that would occur upon removal from the skull.

Removal of the brain and immersion fixation causes microscopic damage

In addition to macroscopic damage, microscopic damage occurs as well. One of the artifacts associated with removal of the brain from the skull are compacted neurons:

Dark neurons; image source

Compacted neurons are an important topic in brain preservation. Here is some more information about them (Csordás et al., 2003):

• They are often called “dark” neurons because they stain dark color in response to certain dyes, including being hyperbasophilic on H&E. This is a result of their constituent biomolecules developing excess negative charges (Gallyas et al., 2009).
• They have a shrunken cell body, a shruken nucleus, and they have slender, shrunken, and “cork-screw”-like dendrites (J. Cammermeyer, 1978).
• Compacted neurons have been called the most common artifact found in fixed brain tissue (Garman, 2011b)
• They can occur as a result of immersion fixation and imperfect perfusion fixation. The only way to prevent them is via perfusion fixation starting at the moment of legal death and allowing for fixation to occur in the brain for at least 1 day prior to removal of the brain from the skull (Csordás et al., 2003).
• They primarily occur as a result of mechanical trauma, such as would occur when removing the brain from the skull prior to fixation or in adjacent regions found near cuts in the brain (Jan Cammermeyer, 1975)
• During perfusion fixation, dark neurons can also occur in transitional zones between white and grey matter, possibly due to a pressure difference generated between these regions as a result of differential perfusate flow to these two regions (J. Cammermeyer, 1978).
• Their distribution can appear haphazard - the compaction phenemenon certainly does not occur to all neurons (J. Cammermeyer, 1978)
• Aside from preservation procedures, they can be caused by a variety of conditions, including ischemia, low blood sugar, status epilepticus, electrical injury, and mechanical injury (Gallyas et al., 2009). Compacted neurons seen following a brain preservation procedure have a similar morphology to those formed during life as a result of these conditions.
• The transition from normal to compacted neuron happens throughout the soma-dendrite compartment as an all-or-nothing phenomenon (Gallyas et al., 2009). It can also occur in axons (Pál et al., 2014).
• The transition from normal neuron to compacted neuron occurs rapidly, in less than 60 seconds (Gallyas et al., 2009).
• Compacted neurons shrink to a significant degree. The mechanism seems to involve rapid loss of water (perhaps up to 50% of cellular water) and gel-gel phase transition. The gel-gel phase transition may help to cause of the dehydration and vice versa (Kovács et al., 2007). This mechanism makes sense to me and I find the evidence convincing.
• They are often surrounded by astrocytes with swollen processes that have likely taken up the fluid from the compacted neurons (Gallyas et al., 2009).
• Dark neurons can still be seen after typical time periods of postmortem autolysis in brain banking, at least up to 24-72 hour duration (J. Cammermeyer, 1979)
• The formation of compacted neurons is reversible. Indeed, some data suggests that reversibility is the expected outcome, assuming that the underlying cause has resolved. In one rat study, the “overwhelming majority” of neurons that were compacted by external forces recovered normal morphology within 4 hours (Csordás et al., 2003). Compacted neurons can also be reversed with proteinase K treatment of fixed tissue sections.
• Under electron microscopy, compacted neurons doesn’t seem to involve damage to cells. Instead, they seem to be due to a “dramatic compaction of apparently undamaged ultrastructural elements” without inducing apparent discontinuities of the plasma membrane (Kovács et al., 2007). Synapses are still present (Gallyas et al., 2009).

Another important artifact that has been described in immersion fixed compared to perfusion fixed tissue is spherical densities (Routtenberg et al., 1974). Spherical densities are likely due to the aggregation of intracellular biomolecules and can occur at synapses. They are also known as nematosomes (Beux, 1973).

Buscaino bodies are another important brain fixation artifact (Wohlsein et al., 2013):

Buscaino bodies or “mucocytes” represent glassy, pale, gray-blue, metachromatic, often PAS-positive, rounded, oval, or lobulated, mucin-like material of approximately 100 μm in diameter that are usually dispersed in the neuropil but preferentially located in the white matter. The exact nature and development of these structures are not known, but they are thought to represent alterations of myelin fixation with solubilization and subsequent precipitation, as well as the handling of nervous tissues too soon after perfusion (i.e., before the chemical process of fixation has been completed). Buscaino bodies have to be differentiated from corpora amylacea by neuroanatomical distribution and at least partial birefringence under polarized light, edema, or material accumulated in storage disorders.

Other cellular artifacts in immersion fixed as compared to perfusion fixed brains include:

• Astrocytes with swollen nuclei, swollen processes, retraction spaces, and aggregations of vacuoles (Garman, 2011b)
• “Fried-egg” appearing oligodendrocytes as a result of a lack of staining in the cytoplasm (García-Cabezas et al., 2016), which may be due to an accumulation of cytoplasmic fluid. Historically, it has been suggested that the accumulation of cytosplasmic fluid in oligodendrocytes can be reversible (Del Bigio et al., 2000)
• Swelling or shrinkage of cells (Koenig et al., 1952)

Many of these artifacts that occur in immersion fixation are also autolytic findings occurring during the postmortem interval (Krassner et al., 2023). So they almost certainly come about because the postmortem interval prior to fixation of the tissue is longer. My personal opinion is that it’s not clear to me that any of these artifacts are going to irreversibly damage the information content of the biomolecule-annotated connectome. If you disagree, I’d like to hear why.

Immersion can be difficult for chemicals that preserve the outer surface quickly

One theoretical concern with immersion-based preservation is that preservation of the outer surface areas can make it more difficult for the preservative chemical to penetrate into the deeper areas. This is often reported with especially strong crosslinking fixatives such as glutaraldehyde (Gerrits et al., 1996). As a result, glutaraldehyde is said to be only useful for preserving small sections of tissue via immersion, such as those 1-2 mm^3 in size. It is unclear how much this matters in practice, and if I recall correctly, I have read some sources stating it is not a major factor, especially with formaldehyde.

How long does the basic method of immersion fixation take to preserve the whole brain?

One study finds that after 24 hours of immersion fixation with 20% formalin at room temperature, human brains tend to stil be unfixed in the center areas, while the outer areas are well-fixed (Scott et al., 2013).

Itoyama 1980 reports that it takes 6-10 days for the central white matter to be fixed via whole brain immersion fixation (Itoyama et al., 1980):

In paraffin-embedded human tissue, whatever alterations occur between death and autopsy continue until formalin has been present for at least 24 hours in the region to be studied. If the whole brain is immersed (as in this study), the central cerebral white matter is not adequately fixed for six to ten days.

An ex vivo neuroimaging study on a human brain hemisphere presents some data on this question (Dawe et al., 2009). They report it takes at least 20 days for paraformaldehyde to reach the center of the brain at refrigerator temperatures, and that even after then, decomposition may still continue.

By the time the first postmortem scan was conducted for any of the hemispheres in this study, the T2 values near the surface of the hemispheres had already decreased to a plateau of approximately 50 ms. Although the decrease was not captured in these experiments, the in vivo value for cortical gray matter is known to be approximately 100 ms at 3T (32). The decrease was apparently due to formaldehyde-induced protein cross-linking, which increases tissue rigidity and decreases T2 values. These results suggest that the process of fixation and its effects on T2 values of near-surface brain tissue are completed relatively rapidly, occurring within a few days of immersion in formaldehyde solution.

In deep tissue, a reduction in the T2 values was observed in the first 20 days postmortem, in all five hemispheres. This decrease was presumably due to the formaldehyde reaching and gradually fixing that tissue. The fact that the decrease in T2 values was slower in deep tissue compared to tissue near the surface of the hemispheres may be due to the fact that a longer period of time is required for formaldehyde to diffuse to the middle portions of the brain and accumulate in sufficient quantities to cause complete fixation. In a 1941 study (26), 30 h were required for 4% formaldehyde solution to penetrate 30 mL into coagulated chicken plasma. In a 1960 study (27), it was claimed that 3 days are required for the same solution to penetrate 30 mL into rabbit liver tissue. Recent findings suggest that approximately 124 days are required for 4% formaldehyde to penetrate 30 mL into whole human spleens (28). Another study found that 62 days are required for 30 mm of penetration into mammalian liver tissue (25). To our knowledge, this is the first study to report that at least 20 days are required for a sufficient amount of formaldehyde to diffuse to the innermost portions of a human brain hemisphere to cause fixation

An ex vivo neuroimaging study of a marmoset brain presents some data (Haga et al., 2019). They find that fixation is pretty fast, but marmoset brains are much smaller than human brains – on average around 3 cm in length and 2 cm wide (Woodward et al., 2018). They report (Haga et al., 2019):

Figure 4 shows the relationship between the values and the postmortem duration in the preliminary examination plotted using a line graph. The T1 values in these regions decreased remarkably within 3 days after tissue fixation and then showed a gradual decrease (Fig. 4a). The T2 and T∗2 values in these regions decreased remarkably within 1 week after tissue fixation and then showed a gradual decrease similar to the trend seen in T1 values (Fig. 4b–4c). The AD, RD, and MD values in these regions showed a large decrease immediately after tissue fixation and remained approximately constant thereafter (Fig. 4e–4g). Regarding FA values, a significant change was not observed in these regions during the observation period (Fig. 4d).

Another study of immersion fixation of whole human brains as measured by MRI scans noted that autolysis was not marked on histology in the deep white matter after the procedure (Shatil et al., 2018):

Two whole, neurologically-healthy human brains were immersed in 10% formalin solution and scanned at 13 time points between 0 and 1,032 h. Whole-brain maps of longitudinal (T1) and transverse (T2) relaxation times, FA, MD, and MWF were generated at each time point to illustrate spatiotemporal changes, and region-of-interest analyses were then performed in eight brain structures to quantify temporal changes with progressive fixation. Results: Although neither of the diffusion measures (FA nor MD) showed significant changes as a function of formalin fixation time, both T1 and T2-relaxation times significantly decreased, and MWF estimates significantly increased with progressive fixation until (and likely beyond) our final measurements were taken at 1,032 h.

It is worth noting that Eriochrome Cyanine-stained tissue sections obtained from each brain after the final, 1,032 h MRI scans appear to show: (1) little, if any, evidence of generalized autolysis and (2) large numbers of myelinated axons within the deep WM (Figure 3). The histology data, therefore, suggest that both the tissue (in general) and myelin (in particular) were reasonably well-preserved—or at least not markedly degenerated—during the course of our MRI experiments.

One source notes that fixation is not “complete” for 5.4 weeks (Yong-Hing et al., 2005). However, it’s unclear what “complete” means in a brain preservation context.

Taken together, there are various ways of measuring this, each yielding different answers. The length of time for fixation also depends on several factors, such as whether the brain is refrigerated or not. More study is required to better model this process.

Variations on immersion preservation

Most of the variations of immersion preservation I will focus on are about improving the speed with which chemicals reach the target tissue.

Convection-enhanced delivery

There are three major ways that chemicals can be transported:

• Diffusion – defined as the spontaneous movement of any material from where it is to where it is not
• Migration – the movement of charged particles in an electric field
• Convection – movement of material contained within a volume element of stirred (hydrodynamic) solution

Most immersion protocols rely upon diffusion, but convection is an enticing possibility to speed up the process. In convection-enhanced delivery, a needle is inserted into brain tissue and chemicals are delivered into the extracellular space at specified flow rates via convection (Casanova et al., 2014). Convection-enhanced delivery is used in neurosurgery to deliver chemicals to the brain. Here is an example of a convection-enhanced delivery system:

Example of convection enhanced delivery system for a rat brain; image source

Convection-enhanced delivery includes manual needle injections into the brain as a special case where the pressure gradient is generated by hand. However, after the initial injection, that approach will primarily rely on diffusion within the brain for the preservative chemicals to distribute.

Convection-enhanced delivery will clearly cause local damage to the brain tissue where the needle penetrates. It also has the potential to cause flow-induced and osmotic concentration damage to the brain tissue where the preservative chemicals leave the needle tip.

The question is whether these sources of damage are outweighed by the upside of faster preservative chemical delivery. In some cases, my guess is that the answer would be yes, but this has not been shown definitively to my knowledge.

Injection of glycerol and ethanol into the brain parenchyma has been used to preserve sheep brains. This study reported injecting the preservative chemicals “in several places—in both the left and right cerebral hemispheres”.

Another factor to consider is backflow of preservative chemical along the needle track. Backflow is often a significant problem in neurosurgery applications, where they are hoping to achieve delivery to a specific tissue area, but could be a benefit in brain preservation. By slowing inserting the needle while distributing preservative chemicals via convection, this could theoretically be intentionally leveraged to distribute the chemical to a larger area of the brain.

One of the challenges with this approach is that it may be more difficult to control an osmolarity ramp of the preservative chemical. This is certainly a problem when using cryoprotective agents, the concentration of which generally needed to be raised in a graded manner to avoid damage due to osmotic concentration differences.

Convection-enhanced delivery of preservative chemicals into the ventricles is a particularly appealing approach. Injecting preservative chemicals into the ventricles is a common approach in postmortem preservation. For example, it was used by the famous German anatomist Walter Thiel (Brenner, 2014). This takes advantage of the extensive ventricular spaces (Shen, 2018) of the brain, including perivascular spaces, to dramatically increase the surface area:

CSF circulation and CSF compartments show the ventricular spaces in the brain; image source

In preservation of the liver, fixation via injection is often called “injection perfusion” (Wisse et al., 2010):

The tissue is transported to an annex to the operation theater, where injection perfusion with glutaraldehyde fixative is performed in a Petri dish filled with saline; (3) The wedge biopsy is held at a corner by a forceps and is injected by a 25 G syringe from multiple sides with 1.5% glutaraldehyde fixative, until a discoloration and hardening of the tissue, comparable to total liver perfusion, is obtained; (4) Care should be taken that the injection needle does not operate as a biopsy needle; it should be and stay completely filled with glutaraldehyde during injection into the tissue; (5) After injecting the needle into the tissue, parallel to the capsule rim, the needle should be withdrawn a few millimeters before starting injection of the fixative fluid. The idea is to create a space within the tissue in which the fixative can spread out; (6) Injections should be repeated several times until the blood is flushed out of the tissue and the consistency has changed to that of a hard-boiled egg Wisse E, Braet F, Duimel H, Vreuls C, Koek G, Olde Damink SW, van den Broek MA, De Geest B, Dejong CH, Tateno C, Frederik P. Fixation methods for electron microscopy of human and other liver. World J Gastroenterol 2010; 16(23): 2851-2866 [PMID: 20556830 DOI: 10.3748/wjg.v16.i23.2851]

Convection-enhanced delivery into the ventricles blurs the line between immersion and perfusion; in fact, sometimes it has been called perfusion. For example, (Toga et al., 1994) describe “perfusion” into the intraventricular space for distributing fixatives in human brains and found that it was more effective for their purposes than perfusion through the blood vessels:

We tested 2 head fixation protocols. In the first protocol, the head was perfused through the common carotid arteries by a constant pressure system (120 mm Hg). The vasculature was flushed with normal saline (4 1) followed by 8% neutral buffered formalin (4-8 1) and the specimen postfixed in 8% formalin for several days to ensure thorough fixation. In the second protocol, the cerebral intraventricular space was directly accessed by metal cranial shunts into one occipital horn and both lateral ventricles anteriorly. Shunts were fixed in place with dental cement and the ventricular system perfused for 72 h with a slow flow of formalin fixative during immersion fixation of the entire specimen…

We assessed several methods for fixative delivery. In the isolated head preparation, vascular perfusion through the carotid and vertebral arteries using a constant pressure system resulted in uneven fixation in several specimens. Erratic vascular clot formation in the postmortem interval may contribute to this non-homogeneity. We obtained good results using intraventricular cannulae for fixative delivery to the brain while the entire specimen was being immersion fixed for several days in a tank of buffered formalin.

Injection of osmium tetroxide into the fourth ventricle was used in the 1950s by Sanford Palay and found to lead to “satisfactory fixation” of the medulla oblongata and the cerebellar cortex as measured by electron microscopy (Palay et al., 1955).

In a brain banking study I was involved in, we injected fixative into the third and fourth ventricle via syringe to speed the rate of fixative delivery (McKenzie et al., 2022).

Ventricular injection has been found to be helpful for preservation in Miyake et al 2020 (Miyake et al., 2020).

Ventricular injection of fixatives in situ through the fontantelle using a lumbar needle attached to a syringe has been reported in infants, with good effect (Bass et al., 1993).

Another abstract reports using subarachnoid injection for brain preservation (Cimmino et al., 2002).

One study reports using injection into subarachnoid space to increase the speed of fixation for fetal brains (Nicholls, 1988):

Once the anterior and posterior fontanelles are exposed a 1 to 2 mm nick is made with a No 15 scalpel blade in the anterior fontanelle 2 to 3 mm lateral to the midline… A new or used epidural catheter is inserted into this neck and fed medially and anteriorly until contact with the base of the cranium is rached. The subarachnoid space is then perfused with Heidenhain’s Susa fixative.

Ventricular perfusion has also been used with good reported efficacy in many animal studies, including one, (Rodríguez, 1969), that studied toads.

Does ventricular perfusion bypass the blood-brain barrier? According to (Gardiner, 1965), it seems to.

Ventricular perfusion seems particularly valuable for preserving periventricular structures such as the thalamus that might not otherwise be well-preserved by standard immersion preservation.

One challenge with ventricular injection is that ventricles lose volume rapidly when CSF is lost in the postmortem period, for example following separation of the head (de Guzman et al., 2016). This might make them more difficult to inject into, although it has been reported to be possible.

In ex vivo samples, ventricles, which are normally filled with cerebrospinal fluid, lose rigidity due to decapitation. Unlike the white and grey matter, there is very little substance inside the ventricles to help maintain their shape

The use of injections of into brain parenchyma has been frequently discussed in cryonics (eg here, here, here, and here although it has not yet been used as far as I know.

Injection is commonly used in biological tissue specimen preparation for large specimens in natural history collections.

Perfusion of the fixative into the tissues by injection through the specimens circulatory system is the most efficient method for fixation of tissues. In practice, however, perfusion is rarely used in the field due to the time and equipment needed and because it requires cutting into the specimen to access the blood vessels and the loss of blood from the specimen. In field practice, smaller organisms are submerged in fixative solutions; larger specimens are injected with fixative solutions. Historically, before the availability of reliable syringes, cuts were made through the specimens to allow the penetration of fixatives.

It’s good to get insights from practical fields. In taxidermy they tend to use formalin injection via hypodermic needles (see here and here):

Find a well-ventilated area (outdoors is easiest) and ALWAYS wear a respirator. Using nonporous, hypoallergenic powder-free nitrile gloves and a puppy pee pad with a plastic backing, which absorbs extra fluid and doesn’t allow it to leak through, prepare your work station. You’ll need a hypodermic needle, a syringe, and a fixative, typically formalin but I’ll discuss other options further down the page. Inject the fixative into the entire specimen - the mouth, through the ears, if the eyes are going to be closed inject the eyeballs, the body cavity, through the anus, into all the large muscles including the ones in limbs. You want the entire specimen to be filled and bloated-looking with fixative. For larger specimens with hair, you can make tiny incisions in the skin to allow the fixative to soak in (explained in the next paragraph). The goal is to get the whole specimen, including under the skin, as filled with fixative as possible.

Changing the volume of fixative to tissue ratio

Many sources point out that in immersion, it is critical to have a high ratio of fixative to tissue ratio. For the human brain, this would require a large amount. For example, per (Høyer et al., 1991):

For fixation by immersion the sample should be placed in at least 20 times its own volume of fixative. If the sample is small enough and the interval between removal and immersion is minimal, the results are usually excellent. For most pathology departments this will be the only practical possibility.

How helpful is this really? The article “How much formalin is enough to fix tissues?” argues that actually there is not much data supporting this (Buesa et al., 2012): It also contains this interesting quote, which seems relevant to the high-fat brain:

It is also worth noting that the water contents of the 3 tissues with similar good results, is higher than for fat and skin (Table 2) this probably influencing the results given that all slices were of the same thickness (2.8 to 3.2 mm) and were equally fixed and processed simultaneously for each volume and time combination. Additionally, NBF penetration of fat is hindered by the insolubility of fat in water.

Another source reports (Steicke et al., 2018):

We used 1:5 as the ratio of tissue to fixative volume. Researchers generally recommend a ratio of tissue to fixative volume around 1:10 or 1:20. However, this recommendation results from personal preferences without specific scientific evidence. Williams et al. have reported the effects of fixation on immunohistochemistry procedures and concluded that there were no differences in the results obtained after fixing human tonsils with formalin at tissue to fixative volume ratios between 1:1 and 1:20. Buesa and Peshkov investigated the tissue to fixative volume required for quality fixation using formalin. They reported that the fixation quality was not effected by different tissue to fixative volume ratios such as 1:1, 1:2, 1:5, and 1:10. Therefore, we believe the tissue to fixative volume ratio of 1:5 we used was appropriate.

More generally, there are a lot of myths and lore in the preservation field. One must constantly be questioning the quality of evidence supporting a given claim. Of course, I encourage you to do that for all of my claims.

Cutting into brain tissue

If the problem with immersion is lack of surface access for preservative chemicals, then a simple approach is to cut into the tissue to increase the surface area. This follows the surgical motto: “to cut is to cure”.

Of course, cutting into brain tissue will cause significant damage at the cut areas. Similar to convection-enhanced delivery, this known damage needs to be balanced against the damage that cuts will prevent due to accelerated immersion preservation.

In human brain banking, the brain is often cut at the midline into hemispheres, so that one hemisphere can be frozen for studies that require “fresh” tissue and the other hemisphere can be fixed for studies that benefit from that.

I have spoken with one investigator in human brain banking who said that they had tried perfusion fixation of single brain hemispheres, but found it to not be much better than immersion fixation. The reason they thought this was the case is that once you get access to the lateral ventricles via cutting the brain at the midline, diffusion is much faster and perfusion does not help as much.

Consistent with this, one study reported that severing the dorsal corpus callosum facilitated entry of the preservative chemical formalin into the ventricular system and significantly sped up the immersion preservation process (Heinsen et al., 2000):

Grape-like widened perivascular spaces (Fig. 8) could indicate the presence of gas-forming bacterias which were not inactivated by the low concentration of formalin and which used vessels as an entrance to deeper unfixed brain parts. On the other hand, high concentrations of formalin will harden superficial (cortical) parts of the brain, thus preventing diffusion of formalin into deeper (central medullary) parts of the brain. Therefore, we used a lower concentration of formalin (10%) and we severed the dorsal parts of the corpus callosum to facilitate entry of formalin into the ventricular system.

Another study reports that making a sagittal cut into the corpus callosum allows for the preservative chemical to replace the cerebrospinal fluid (CSF) (O’Sullivan et al., 2019):

An in situ post-mortem image is indispensable for comparison of either 3 T or 7 T MR images and histology. Figure 1b documents the same brain after formalin fixation. In general, the resolution of a 7 T scanner is far superior to a 3 T scanner. However, fixation artefacts by formalin cause a nutshell-phenomenon (Fig. 2b). Initially, formalin rapidly penetrates human central nervous tissue. After a few millimetres fixation-induced cross-linking of proteins imposes a kind of barrier that impedes a further penetration of formalin into the deepest parts of the brain. Early opening of the telencephalic ventricular system by a sagittal cut into the corpus callosum facilitates the replacement of cerebrospinal fluid (CSF) by formalin with a concomitant fixation of external and internal brain parts.

Severing the dorsal corpus callosum would clearly lead to damage in those white matter tracks, but it would lead to the diffusion speed advantages of brain hemisection without having to cut the entire brain in two pieces. In life, severing the brain at the midline is a rare neurosurgery procedure called a corpus callosotomy used to treat severe epilepsy. It can lead to “split brain syndrome”, so it clearly has severe effects on personality, but this seems to be due to the lack of communication between the two hemispheres, rather than a loss of information as a result of the cut itself. It seems likely to me that damage from cutting at the corpus callosum could be repaired with future inference technology.

To be more precise, let’s consider what type of damage would occur with a sectioning procedure and how the original state might be inferred.

If the cut is made in the white matter, then the major structure that will be damaged in the biomolecule annotated connectome are myelinated axons. Axons branch a significant amount, so a good fraction of neurites will cross any given cut interface (examples of axon branching in (Li et al., 2018).

Axons on two sides of a cut interface can theoretically be stitched together. High-quality stitching between cell processes has already been performed across cut brain tissue slices (Xu et al., 2021). These cuts were made in embedded tissue, which means that the cuts are more precise than if they were made in fresh tissue, but I believe the general principle will hold true, especially as our inference and stitching technology improves. A theoretical future solution to the stitching problem will also be significantly aided by the fact that neurites express a unique set of molecules, such as protocadherin cell surface molecules (Brasch et al., 2019)

If the cut were present in the grey matter, then there would be more direct damage to neurons that may directly encoded memories and other aspects of personal identity. Inferring the original state will likely be more difficult and more information loss should be expected.

Another thing to consider is that in neurosurgery, cuts are made in the brain parenchyma, including the grey matter, all the time. On the one hand, neurosurgery obviously causes damage. As the saying goes, “you ain’t never the same, once the air hits your brain.” On the other hand, loss of personal identity is not a typical part of the informed consent procedure in a neurosurgical procedure. The damage is usually relatively limited.

Cutting fresh brain tissue is difficult as the brain is quite mechanically compressible. A common description of its texture is tofu. Imagine trying to cut a large piece of tofu. Not the easiest thing in the world.

It might make sense to let a preservative chemical such as formaldehyde diffuse into the whole brain for 30 mins - 3 hours of so prior to cutting the rest of the brain. Cutting is also a lot easier if the brain is cooled to refrigerator temperature.

One could imagine performing more cuts into the brain tissue than just one at the corpus callosum. Each cut would lead to more damage at the cut interface and more risk of acceleration-based damage in the interior of the brain tissue, but increase the surface area and speed of preservative diffusion into the brain tissue.

Theoretically, one could imagine a radical approach that would cut the brain tissue into sections small enough (500 μm) that they have been shown to retain viability via immersion cryoprotection and vitrification (Pichugin et al., 2006). However, this would require an inordinate number of cuts, perhaps around 300 cuts in the coronal plane (15 cm / 500 um) and 4 in the saggital plane. In practice, this would be enormously technically challenging and cause a significant amount of damage at and nearby the cut interfaces. Attempting this radical amount of cutting seems likely to cause information-theoretic death on its own.

Examples of damage from cutting into brain tissue: (Garman, 2011a):

Where saws (either oscillating electric or manual) are used to remove brains from the larger animal species, contact between the saw blade and the brain surface produces sharp lines of neuropil displacement/compression accompanied by deposition of bone fragments, mineralized debris, hair, or other structures within the brain. Clefts in the neuropil (e.g., Figure 3B) are easy to produce in fresh or freshly fixed brains by even light pressure

Overall, slicing the brain tissue into many pieces prior to the initial preservation seems to be worse than convection-enhanced delivery approaches or other ways of speeding up immersion preservation. Although a smaller number of targeted cuts, such as one in the corpus callosum, is potentially worthy of consideration and modeling.

Immersion via burr holes or skull windows

Immersion methods generally involve removal of the brain from the skull. This is one of the major downsides, as the brain extraction procedure is technically challenging, necessarily causes damage in the cranial nerves and brainstem/spinal cord, and can cause movement-associated damage in the interior of the brain.

However, extracting the brain is not necessarily required for immersion. One could also immerse the brain in preservatives while it is still in its original position, if part of the skull and dura can be removed. This opening can be called a burr hole, skull window, or craniectomy:

Image source

Immersion-based preservation via a burr hole is sometimes done in brain banking. For example, in one protocol, a 6 mm hole is drilled into the skull and a needle is placed through the dura mater into the brain tissue prior to the injection of formalin (a preservative chemical) (Crosado et al., 2020).

One could imagine creating multiple burr holes in order to allow access from multiple parts of the brain. For example, one study performed immersion-based preservation (following perfusion-based preservation) of mouse brains by creating multiple perforations of the skull in a “sieve-like way” and then allowing formalin to diffuse into the brain for at least 3 weeks (Quester et al., 2002). They found that this “effectively optimize[d] infilitration of the complete skull-brain specimen with formalin.”

One study in minke whales created a skull window and then poured fixative into the skull window for immersion fixation (Knudsen et al., 2002). The brain was only removed from the brain once it was fixed. They reportedly relatively good success with this technique, despite the large size of the whale brain. Chana Phaedra notes that this study speaks favorably to the prospects for immersion fixation in human preservation cases.

Combined with convection enhanced delivery, this could allow rapid immersion-based preservation throughout the surfaces of the brain without the problems involved in removal of the brain from the skull.

Using a higher concentration of preservative chemical

Using higher concentrations of preservative chemical will speed the immersion procedure. A higher concentration gradient of the preservative chemical should increase its rate of diffusion into the tissue. This makes basic biophysical sense due to Fick’s law of diffusion.

For example, one study found that 10% formalin led to a faster fixation rate in pig hearts than 2% or 4%, at least at the longest time interval studied (168 hours) (Hołda et al., 2017). If 10% is faster than 2% or 4%, then 20% or 30% formalin would likely be faster than 10%.

Theoretically, a higher concentration of fixative could also lead to faster fixation of the surface layers. This would then inhibit the diffusion speed of the preservative chemicals. But I doubt that this is a major effect.

Dilute to concentrated fixative

It’s also possible to imagine altering the concentration of the fixative during the procedure. (Routtenberg et al., 1974) used an immersion method where the fixative concentration was first dilute and after 2-4 hours was switched to a more concentrated formulation, presumably so that the dilute fixative would initially penetrate better into the tissue:

For immersion fixation of anesthetized animals, subjects were decapitated and the brain was rapidly dissected from the cranial cavity. Frontal sections 3-4 mm thick were made with a clean razor blade and placed in lo-20 times their volume of cold (5°C) dilute fixative recommended by Karnovsky (1965) and Peters (1970). 90-120 set elapsed from the time of decapitation to immersion of the frontal sections in fixative. After 24 hr in the dilute aldehyde, the tissue was placed in a concentrated fixative (Karnovsky, 1965 ; Peters, 1970) at 5°C for 1 hr.

(The same group also used a dilute to concentrate fixative switch during perfusion fixation.)

Superfusion - shaking or stirring the fixative continuously

Superfusion is the continuous flowing of a fluid outside of an isolated organ. This will increase the speed of diffusion in an analogous way to convection-enhanced delivery: it delivers a pressure gradient. But instead of being inside of the brain, the pressure gradient is at the surface.

Superfusion could be supplied by shaking the fluid, stirring it, dripping it, or using tubing to create a laminar flow. The optimal rate of shaking or stirring is to be determined 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 translational acceleration.

This approach makes biophysical sense and it has already been shown to significantly increase fixation speed in freeze substitution (Reipert et al., 2018). So it should also be helpful at above-zero temperatures as well.

In the 1950s, dripping chilled osmium at 4xC onto the surface of the brain for preservation of the cortical surface of rat brains was found to lead to good results (R. L. Schultz et al., 1957).

Removal of meningeal layers

Most articles report that the dura needs to be removed for adequate immersion fixation. One source reports that in order to adequately immersion fixation of part of the brain with osmium, the speed of preservation was also increased if the pia was removed from the cortical surface with forceps (R. L. Schultz et al., 1957). It’s unclear if this would generalize to other preservative chemicals.

With adult rats adequate fixation could be achieved only by stripping the pia from the cortical surface with fine forceps. In young rats this procedure was not absolutely necessary, but it was desirable.

One other source reports that pia removal can improve immersion-based preservation (Pallie et al., 1958). Whether removing the pia is actually helpful, or worth the time and procedural complexity, seems to be an open question. To my understanding, while removing the dura is essential, removing the pia is not commonly performed.

Optimizing the preservative mixture to increase diffusion speed

Another approach that can be used in immersion preservation is to use a mixture of chemicals, with the goal that some chemicals help increase the penetration speed of the others.

A classic example is DMSO. DMSO helps to increase the rate of penetration of other chemical agents. It allows other chemicals, such as fixatives, to more rapidly penetrate through the interstitial areas of tissue (i.e., the areas between cells) (Malinin et al., 2004). DMSO has been decreased as a “well-known skin penetration enhancer” (Rylander et al., 2006).

A key question is the trade-off to the use of DMSO: to what extend does DMSO immersion damage tissue elements, which might outweigh the increased preservative chemical diffusion speed? There is some evidence that DMSO can cause damage to cellular structures, such as causing plasma membrane damage and dissolution of the cytoskeleton. However, it is also a commonly used cryoprotectant with a reasonable safety profile in that context. Its effects on cells are likely also different in combination with fixatives. The effect of DMSO strongly depends on the concentration used (Malinin et al., 2004).

Another chemical that has been used to increase diffusion speeds is hyaluronidase. Hyaluronidase treatment has been used to accelerate immersion preservation with osmium (Pallie et al., 1958). Hyaluronidase can be used to cause breakdown of the extracellular matrix in the brain (Susaki et al., 2020) (Soria et al., 2020). Of course, this will also cause damage to the extracellular matrix, which could cause information loss. Although in my opinion, most likely not an uninferable information loss as long as other aspects of the tissue microenvironment and breakdown products are preserved. Still, the damage caused by hyaluronidase treatment makes it likely not the best option for this.

The use of the detergent saponin has been reported to improve immersion preservation of brain white matter with osmium (Luse, 1960). This might lead to damage to tissue lipids and cell membranes, though. Saponin has also been used in combination with glutaraldehyde for perfusion-based preservation (Fukudome et al., 1992).

Adding 5% ethanol has also been found to increase the penetration speed of formalin (Nazemorroaya et al., 2022).

There are numerous other possible chemicals that could be used as adjuvants to improve the diffusion of preservative chemicals into brain tissue, each of which might cause different types of damage. Evaluating the net effect of diffusion accelerators will require empirical assessments.

Optimizing the preservative mixture to balance diffusion speed vs preservation trade-offs

In addition to using adjuvant chemicals to increase diffusion speed of primary preservative chemicals, it is also possible to use multiple preservative chemicals, each with different diffusion speeds and preservation quality properties.

The basic idea here is that chemicals that preserve relatively worse but diffuse faster can provide initial stabilization of the tissue before the chemicals that diffuse relatively slower but preserve relatively better can reach the tissue.

Sodium azide is one example of such an adjuvant chemical. Sodium azide diffuses better than glutaraldehyde due to its lower molecular weight. It acts to quickly to inhibit the enzyme ATPase, thus halting mitochondrial activity and acting as a metabolic poison (Minassian et al., 1979).

One study used sodium azide in the immersion preservation of 3 mm blocks of mouse liver (Minassian et al., 1979). They found that adding sodium azide to glutaraldehyde led to better preservation quality in the deep areas of the liver, as evaluated by electron microscopy, especially of mitochondria. Another study found that adding sodium azide to glutaraldehyde improved preservation of mitochondria structure in the lens (Gorthy et al., 1984).

Formaldehyde diffuses much faster than glutaraldehyde, so sodium azide might not be as helpful when used in combination with formaldehyde. It’s also important to point out that sodium azide is also a severe poison to humans. For this reason, its use needs to be minimized and any toxic dose avoided if possible.

Commercial formalin is a mixture of formaldehyde and methanol. Methanol is generally used to decrease the polymerization of formaldehyde during storage. But because methanol seems to diffuse more rapidly than formaldehyde, it may actually lead to a degree of initial coagulative fixation-based preservation, followed by the slower formaldehyde cross-linking fixation (Steicke et al., 2018). The small concentration of methanol in formalin also may cause damage to tissue biomolecules by permeabilizing cells and/or dehydration. To my knowledge, the practical effect of methanol in formalin immersion preservation is not well characterized.

Refrigeration during immersion preservation

Some studies suggest increasing the temperature of the tissue to be preserved, which increases the diffusion speed. But this also increases the rate of decomposition. In general, the increased rate of decomposition seems to clearly outweigh the benefits of increased diffusion speed from a brain preservation perspective. Instead, the opposite is more commonly recommended, that the brain should be refrigerated during immersion fixation.

Hoyer 1991 note that decreased temperature causes decreased penetration speed, but the decrease in autolysis rate more than makes up for this (Høyer et al., 1991):

Autolytic processes place an upper limit on optimum temperatures for immersion fixation. lf the temperature is lowered from room temperature to 0-4°C, the combined reduction in speed of fixation and the autolytic process gives acceptable results. It must be stressed that, unless the specimen is very small, fixation at temperatures above 4°C leads to the tissue only being suitable for a rather coarse light microscopic diagnostic assessment. Unfortunately tissue removed at operations is traditionally, but nonetheless detrimentally, fixed at room temperature. Fixation should always take place at 0-4°C for histochemical work. It should be noted, however, that the addition of CPC (cetylpyridinium chloride) to a concentratiön of 0.5% w/v for improved fixation of proteoglycans (Sect.13.4) leads to precipitates in the fixative at temperatures below O°C.

One study compared the preservation of 4 mm thick breast cancer tissue samples in formalin at room temperature and refrigerator temperature (4xC) (Gündisch et al., 2015). They found an increased preservation quality of phosphorylated proteins in the samples preserved via immersion in formalin at refrigerator temperatures.

One study found that there was improved preservation of tissue when it was preserved at 4xC than 37xC using immersion fixation (Bamisi et al., 2020).

One study found that fixation at low temperature led to better preservation of DNA and RNA (Bussolati et al., 2011).

McKee 1999 notes that refrigeration of the brain during immersion fixation is essential in promoting the quality of preservation (McKee, 1999).

A mathematical modeling study by Mike Perry and Aschwin de Wolf finds that immersion fixation of the brain is better performed at refrigerator temperatures than room temperature:

We find however that the decrease in the reaction rates by the Q-10 rule is greater proportionally than that of the diffusion coefficient, so that lower temperatures diminish the S-MIX [AM: a measure of decomposition], down to the lowest practical diffusion temperature, 0°C, for which the calculated S-MIX is about 5 hours versus about 20 hours at 37°C.

Ultrasound-based acceleration of immersion preservation

Ultrasound has been shown to increase the speed of fixation in tissue blocks (Chu et al., 2005). One study found that ultrasound increased delivery speed of non-fixative chemicals (at the end of a catheter) by 2-3x (George K. Lewis et al., 2012). 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 up diffusion directly.

Ultrasound might itself damage cellular morphology and/or biomolecules. However, considering that ultrasound has also been used in vivo, for example in opening the blood-brain barrier, it may be not cause too much damage to brain tissue, at least if the right parameter settings can be identified.

Elevated pressure fixation

An elevated barometric pressure of up to 15,000 PSI has has been reported to accelerate the fixation speed of porcine liver specimens by 5-fold (Chesnick et al., 2010). That article doesn’t really discuss the mechanism, but it might have something to do with tissue cavitation, which gives formalin additional avenues to diffuse within the tissue, as may occur with ultrasound as well.

Increased pressure has also been found to improve antibody penetration (Fiorelli et al., 2020). However, this study’s evidence is preliminary. This is the speculated mechanism:

We hypothesize that the increased barometric pressure favors antibody diffusion through two concerted mechanisms. First, favorable diffusion conditions are formed immediately following initial pressurization. Because of tissue’s high hydrodynamic resistance, a temporary pressure gradient is created between the sample and its surroundings. This differential drives the antibodies into the tissue at an increased rate, relative to nonpressurized conditions, until pressure is equalized. Second, the increased atmospheric pressure generates an increase in the Brownian motion of the antibodies in solution, thus improving mixing. Because the box is a closed system, the added N2 mass during pressurization is trapped within a fixed volume. The overly abundant gas molecules more frequently enter the liquid surrounding the tissue, interacting with the antibodies within the solution. These collisions subsequently increase molecular motion and, as a result, improve the probability that the antibody will enter the tissue.

Elevated pressure does not currently seem to be practical for such a large organ as the human brain. The devices described in these two articles, the Barocycler 2320EXT and Figure 1C in (Fiorelli et al., 2020), are for quite small biospecimens. I haven’t looked into it further but my guess is that finding or building a large enough device to actually elevate the pressure would be the biggest challenge. Human brains are around 5.5 x 6.5 x 3.6 inches, and the device to store them would need to be substantially larger, to include a container with enough formalin.

Comparisons of perfusion to immersion fixation

Comparing perfusion to immersion fixation, Bywater 1962 come out strongly in favor of perfusion (Bywater et al., 1962).

The illustrations published here should testify to the great significance which the type and the time of fixation have for the cytological observation carried out with the light microscope, in particular, the value of perfused material, foreven tissue immediately immersed in fixative still suffers structural deterioration which corresponds to changes seen in an animal perfused after 1 hour, Even if the size of the tissue is reduced to 3mm artefacts occur which resemble those found in material perfused only after 1 hour. On the other hand it must be emphasized that perfusion should always be attempted, and that perfusion 20 minutes after death shows only minute changes and is hardly distinguishable from material perfused immediately (after death). It proves that the changes which must occur within the first 20 minutes are at submicroscopical level for they can be revealed with the electron microscope but are not yet discernible with our ordinary methods of microscopy [silver staining].

Combination methods

Given unlimited resources, preservation via combination perfusion and immersion is likely the best approach to get preservative chemicals into brain tissue. There are a couple of important exceptions:

1. If the perfusion quality is very poor, then the osmotic damage or fluid overload damage it could cause may mean that it is more harmful than helpful. In that case, immersion-only preservation approaches should be considered.

2. If it takes too long to access supplies to perform perfusion, then it may not be worth it to wait, if immersion preservation could instead proceed immediately. Tissue decomposition prior to preservation can be severe. Waiting many hours to perform perfusion may not be worth it vs proceeding with immersion fixation.

3. If perfusion can be verified to have been successful, such as via imaging of the preservative chemicals throughout the brain, then immersion preservation is not necessary.

One practical way to achieve this is to perform perfusion-based preservation with the brain in its original location in the skull. And then once the perfusion is initiated, remove part of the skull to monitor the perfusion quality and begin an immersion preservation procedure at the same time. If the perfusion fails, then one could begin methods to accelerate the immersion preservation.

Discussions of this topic have long been held in cryonics, for example here, here, and here.

Almost every brain banking study I have seen that uses perfusion fixation does subsequent immersion fixation. Even in most animal studies in which perfusion fixation is used at the time of death and is expected to be very high-quality, immersion fixation is used as well. It seems that most investigators do not want to rely on perfusion alone for tissue preservation.

Summary

The question of whether it is preferable to use immersion vs perfusion for delivering preservative chemicals cannot be considered in isolation.

First, it depends heavily on how much damage has occurred to the brain’s vascular system prior to the start of the preservation procedure. However, the question of how much damage the brain can undergo before perfusion is no longer useful, or may even be harmful, is very much unknown, with little hard data to go off of.

Second, it depends on what level of detail of information is required to preserve the brain. If a very precise level of detail is required, then it may be essential to try perfusion, even when the probability of technical success is lower. Even if it does not work, attempting a perfusion may have been the only chance to preserve the information content prior to decomposition. On the other hand, if a less precise level of detail is required, then immersion may be sufficient, and too much focus on perfusion as a necessity may cause increased expense and lack of access for a very large segment of the population. The optimal solution depends in large part on how hard the problem is of preserving engrams and other valued structural information in the brain.

Third, it depends on where in the brain the information for long-term memories and personality styles is primarily present. If the important structural information is primarily present in the approximately 6 mm of the surface of the brain known as the cortical ribbon, then this may preserved by surface-based immersion preservation. On the other hand, if required structural information in inner brains regions would not be sufficiently preserved by standard immersion preservation, then perfusion or effective approaches to speed up immersion may be necessary.

Fourth, it relates to how much damage occurs during the postmortem interval prior to preservation. If a significant amount of damage occurs rapidly during the post-mortem interval, even at refrigerator temperatures, then perfusion may be necessary to preserve the brain tissue prior to decomposition. On the other hand, if the damage is slower, then there is more time to wait for immersion-based approaches to work while the chemicals diffuse in, and perfusion may be relatively less necessary.

Finally, it depends on which preservative chemicals are used. Immersion with cryoprotective agents is generally not considered possible, so immersion is currently thought to be only an option for procedures that use fixatives.

If the level of detail required for sufficient inference of the biomolecule-annotated connectome is not as high, the cortical ribbon is the most important part of the brain, cutting the brain tissue prior to preserving it is acceptable, and/or postmortem decomposition at refrigerator temperatures is relatively slow, then immersion-based approaches have a higher probability of being “good enough.” In this case, poor perfusion methods can instead be thought of as having serious risks, including (a) causing damage to brain tissue due to potential osmotic damage and edema, (b) delaying the start of preservation to wait for the team and equipment necessary for perfusion, thus increasing damage due to postmortem decomposition, and (c) decreasing access to brain preservation due to the technical expertise for perfusion being rare and the cost being high.

Given our uncertainty about all of these questions upon which the perfusion vs immersion decision hinges, a reasonable approach today may be to say that perfusion is the better option and in most cases is worth attempting if possible, but that immersion fixation is also a reasonable option if perfusion cannot be accessed.

Further reading

• History of embalming and human body preservation: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3931544/
• David Bodian’s 1936 article includes numerous tips on brain perfusion: https://onlinelibrary.wiley.com/doi/epdf/10.1002/ar.1090650110
• Mike Darwin on choosing the approach for perfusion-based cryopreservation: http://www.cryonet.org/cgi-bin/dsp.cgi?msg=2922
• Excellent paper on choosing optimal pressure for perfusion fixation: https://www.sciencedirect.com/science/article/pii/S0165027022000206
• Research on cooling rates of the brain, relevant to stabilization: http://www.oregoncryo.com/researchCoolingRates.html

Endnotes

Microwave to speed immersion preservation

As (Murphy, 2010) put it: “To microwave or not to microwave, that is the question that has confounded the neurochemist as the quest for reducing changes in neurochemicals associated with post-mortem delay has evolved over the years.”

(G. R. Login et al., 1988) is one of the first studies to use microwave irradiation to improve immersion fixation on tissue samples. They found that microwave irradiation alone led to some amount of ultrastructural preservation. The best preservation they found was the use of low concentrations of fixatives (formaldehyde and glutaraldehyde) alongside microwave irradiation. The irradiation could be as fast as 26 milliseconds. They reported that allowing the temperature of the sample to get too high, such as greater than 50xC, likely leads to tissue damage. They also reported that microwave irradiation could only be effectively used on smaller tissue samples, as “microwave energy at a frequency of 2.45GHz can penetrate approximately 1.5-2cm into physiological salt solutions and biological specimens.” Thus, biospecimens larger than 1-2 cm in all dimension would be susceptible to uneven warming and associated tissue damage. The average whole human brain has dimensions of 14 cm wide, 16.7 cm long, and 9.3 cm high.

One benefit of temporarily microwaving to temperatures of around 80xC is that it can rapidly inactivates enzymes, decreasing the rate of tissue decomposition. Of course, this also will affect biomolecule conformation, but as discussed in a previous chapter, biomolecule conformation is likely not a unique store of information. Also, this high temperature may also lead to structural damage.

The use of microwave irradiation of human brain hemispheres has been reported to speed the process of immersion fixation with good preservation quality at the light microscopy level at least (Boon et al., 1988).

A good review on the topic (Gary R. Login et al., 1994) makes the following points:

• Microwave stabilization prior to chemical fixation of brain tissue has been shown to lead to worse structural preservation than immersion fixation.
• “[I]f the specimen is located beyond the penetration depth of microwave power, the final morphological result is influenced more by conductive heating than by microwave irradiation” - biospecimens need to be small for microwave irradiation to be useful, need to have at least one dimension less than 1-2 cm.

Another good review on the topic (Kok et al., 1990):

Listed below are the sixteen most frequent misconceptions encountered in the past 5 years during our presentations at conferences and courses: (1) microwaves can only be used in the kitchen, (2) microwaves act only on water content of food, (3) 2.45 GHz is the frequency of a sharp resonance, (4) a magnetron is a microwave oven (widespread in Holland), (5) a ‘hot spot’ is a fixed (hot) spot in the microwave oven, (6) a ‘cold spot’ is a fixed (cold) spot in the microwave oven, (7) the larger the load the slower the rise in temperature, (8) the smaller the load the faster the rise in temperature, (9) the smaller the-load the slower the rise in temperature, (10) the larger the load the faster the rise in temperature, (1 1) fat heats up slowly because it has a small dipole moment, (12) low power level means low power level, (1 3) the displayed temperature is the temperature of the load, (14) a simple kitchen microwave oven cannot be used in a laboratory, (15) the microwave effect can be separated from the temperature effect, (16) microwave methods cannot be used in developing countries.

Microwaves seem to also help chemicals penetrate the brain (Feirabend et al., 1991):

Microwave irradiation may also enhance the effect of a fixative by accelerating its penetration, changing its chemical structure and reinforcing its binding to the tissue proteins. For example, formaldehyde is mainly present in tissue as a non-fixating substance methylene glycol. Microwave irradiation promotes penetration of the latter, its conversion into formaldehyde and binding of this substance to proteins (Walker, 1964; Fox et al.1985). Microwave irradiation may also improve penetration of dyes into the tissue and improve their binding to the tissue components (Boon and Kok, 1989; for brain tissue see Marani, 1989).

One study found that microwaves seem to be helpful in fixation of placental tissue (Henwood, 2018).

The use of microwave-accelerated glutaraldehyde immersion fixation has been shown to help synapse preservation in hippocampal slices, allowing for preservation within seconds (Kirov et al., 1999).

Taken together, the use of microwave-accelerated immersion preservation would likely require either improvements in microwave technology over those available in the 1980s and 1990s and/or the use of sectioning the brain prior to immersion preservation.

Venous perfusion

One possibility that has been reported is venous perfusion. While this is obviously the reverse direction as blood flows during life, the venous system may still be able to carry preservative fluid, so it may not matter that it is not physiologic. Some investigators have reported that venous perfusion works better than arterial perfusion; for example, this was reported by (Hamlyn, 1997).

Vacuum to speed immersion fixation

Another possibility is that a vacuum force could be used to speed up immersion preservation, similar to the use of vacuum infiltration in polymer embedding/plastination. The use of vacuum infiltration has previously been reported for plant tissue; however, there is an obvious concern that the use of vacuum would lead to significant damage to microstructure that would outweigh any benefits of increased immersion speed (Paiva et al., 2011).

Link to the next essay

https://brainpreservation.github.io/

Link to return to the index

https://brainpreservation.github.io/

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