Vitrification agents in cryonics: M22

M22 represents the culmination of decades of work in applied cryobiology by researchers Gregory Fahy , Brian Wowk, and others to develop a vitrification agent that can recover complex organs (such as the kidney) from cryogenic temperatures without ice formation and minimal toxicity. In 2005, M22 was licensed by the patent holder 21st Century Medicine (21CM) to the Alcor Life Extension Foundation to replace their previous vitrification agent B2C. As a result, the least toxic vitrification agent for complex organs that has been documented in peer review journals is currently being used for cryonics patients at Alcor.

M22 incorporates a number of important discoveries in cryobiology:

1. High concentrations of a cryoprotective agent (or a mixture of different cryoprotective agents) can prevent ice formation during cooldown and warming.

2. The toxicity of some cryoprotectants can be neutralized by combining them with other cryoprotective agents.

3. The general toxicity of a vitrification agent can be predicted by using a measure called qv*, allowing for the rational formulation of less toxic vitrification agents.

4. Within limits, non-penetrating agents can reduce the exposure of cells to toxic amounts of cryoprotectants without reducing vitrification ability.

5. Synthetic “ice blockers” can be included in a vitrification mixture to reduce the concentration of toxic cryoprotective agents necessary to achieve vitrification.

6. Substituting methoxyl (-OCH3) for hydroxyl groups (-OH) in conventional cryoprotective agents can decrease viscosity, increase permeability, and reduce the critical cooling rate necessary to avoid ice formation.

7 Chilling injury can be eliminated by introducing the vitrification agent with a hypertonic concentration of non-penetrating solutes.

8. In cryonics, with a minor proprietary modification, M22 can be used for whole body perfusion without causing severe edema that has been a problem for some other solutions.

Vitrification is the solidification of a liquid without crystallization. When a solution is cooled down to the glass transition point (-123.3°C for M22) the extreme elevation in viscosity will produce a glass in which all translational molecular motions are arrested. Although water vitrifies at cooling rates exceeding a million of degrees Celsius per second, such cooling rates are relaxed when other solutes are substituted for water. In cryobiology solutions with high concentrations of cryoprotective agents can be used to vitrify complex organs such as the kidney or the brain.

Vitrification has a number of clear advantages over conventional cryopreservation. The most important advantage is the elimination of ice formation. Although the adverse effects of ice formation can be mitigated by the use of cryoprotective agents (glycerol, DMSO) and optimization of cooling rates, massive ice formation does not permit recovery of complex organs with full viability. Another advantage is that vitrification eliminates the need to strike a balance between the risk of intracellular freezing induced by fast cooling on the one hand, and cell dehydration and solution concentration induced by slow cooling on the other hand.

The challenge in formulating successful cryoprotective agents is to design vitrification solutions that are non-toxic but allow for vitrification at realistic cooling and warming rates. For more than a decade the least toxic vitrification agent was Greg Fahy’s VS41A, which is an 55% weight/volume equimolar mixture of DMSO and formamide plus propylene glycol. The “1A” in VS41A reflects the solution’s ability to vitrify at normal atmosphere pressure (as opposed to an older, more dilute solution, VS4, which requires 1000 atmospheres of pressures to vitrify). The equimolar concentrations of DMSO and formamide reflect Baxter and Lathe’s research who concluded that amides can neutralize the toxicity of DMSO, a finding that Greg Fahy later revised in favor of the theory that it is actually DMSO that neutralizes the toxicity of formamide. The ability of DMSO to neutralize the toxicity of formamide (up to certain concentrations) allows for the formulation of vitrification agents with reduced toxicity. This finding has been so fundamental that an equimolar concentration of DMSO and formamide remains the core of M22.

Another major step was made when the researchers at 21CM found that high concentration of (penetrating) cryoprotectant agents do not necessarily increase toxicity. Contrary to conventional cryobiology expectations, Fahy et al. found that weaker glass formers favor higher viability. They proposed a new compositional variable called qv* to predict the general toxicity of vitrification solutions. Using qv* they made the “counter-intuitive” decision to substitute a higher concentration of the weaker glass former ethylene glycol for propylene glycol to create a solution called Veg, which produced a substantial improvement in terms of viability as measured by K+/Na+ ratios.

Because cells contain higher concentrations of protein, the intracellular space is more favorable to vitrification than the extracellular space. As a consequence, the concentration of penetrating (toxic) cryoprotectants can be reduced in favor of non-penetrating polymers like polyvinylpyrrolidone (PVP). Variations of Veg in which the concentration of DMSO and formamide was reduced in favor of PVP increased viability without decreasing its ability to suppress ice formation. The concentration of penetrating cryoprotectants can be further reduced by inclusion of non-penetrating “ice-blocking” polymers. These ice-blockers also reduce the critical cooling and warming rates necessary to avoid ice formation, which is an important requirement for solutions that are used to vitrify complex organs such as the human brain.

Because concentrated vitrification solutions depress the homogeneous nucleation temperature (Th) below the glass transition temperature (Tg), a major obstacle to successful vitrification is the presence of heterogenous nucleators. Some organisms have antifreeze proteins (AFPs) and anti-freeze glycoproteins (AFGPs) that mitigate heterogenous nucleation by binding to nucleators. Because adding such anti-nucleating proteins to vitrification solutions would be prohibitively expensive and less effective, Greg Fahy proposed the creation of synthetic ice-nucleation inhibiting polymers. In 2000 Wowk et al. published work that showed the effectiveness of a co-polymer of polyvinyl alcohol (PVA) and vinyl acetate in inhibiting heterogenous ice-nucleation. This co-polymer is now being sold by 21CM under the name “X-1000”. X-1000 is particularly effective in glycerol solutions, presumably because glycerol itself is a poor anti-nucleation agent. Increasing the concentration of X-1000 in vitrification solutions decreases ice formation and relaxes minimum cooling rates. Although X-1000 is presumed to be non-toxic, the maximum concentration in vitrification solutions does not exceed 1% w/v because no further benefits were observed beyond this concentration. In 2002, 21CM announced the discovery of another synthetic “ice-blocker” called Z-1000. Z-1000 is the polymer polyglycerol (PGL), which specifically inhibits ice nucleating activity caused by the bacterium Pseudomonas syringae. Mixtures of PVA and PGL are more effective in inhibiting ice formation than either agent alone, suggesting the PVA and PGL complement each other by inhibiting different sources (bacterial and non-bacterial) of ice nucleation.

A variant of Veg that includes the low molecular weight polymer polyvinylpyrrolidone K12, X-1000, and Z-1000 named VM3 improved viability in renal cortical slices and decreased the critical cooling and warming rates necessary to avoid ice formation and de-vitrification (ice formation during rewarming) while maintaining the same molar concentration as VS41A. The transition from Veg to VM3 reflects the two breakthroughs mentioned above: reduction of cryoprotectant toxicity by inclusion of non-penetrating polymers and ice blocking agents. VM3 also was the least toxic agent in vitrification of rat hippocampal brain slices, which is of particular importance for cryonics. The first vitrification agent ever to be introduced to cryonics was a hyperstable variant of VM3 called B2C. B2C was used until late 2005, when it was replaced by M22.

M22 takes advantage of two other discoveries: the ability to design better glass formers by methoxylation of conventional polyols, and inhibition of chilling injury by delivering the vitrification agent as a hypertonic solution. Because hydroxyl groups can bind either to water or hydroxyl groups on other cryoprotective agents, substituting methoxyl groups for hydroxyl groups should decrease interaction between cryoprotectants and increase interaction between the cryoprotectant and water. As a result, methoxylated compounds have stronger ice inhibiting ability, thus reducing the critical cooling rate for vitrification or reduce the concentration of (toxic) cryoprotective agents in a solution. Methoxylated cryoprotectants also decrease viscosity and increase cell permeability, allowing for shorter perfusion times, and thus reduced cryoprotectant exposure at higher temperatures. For example, the methoxylated glycerol derivative 3-methoxy-1,2-propanediol has a higher glass transition point and vitrifies at ~ 5% lower concentration than the corresponding conventional cryoprotective agent. Complete exploitation of these advantages is limited by the fact that they are more toxic than their non-methoxylated compound, as predicted by qv*. As can be seen in the table, the major difference between VM3 and M22 is the reduction of PVP K12 in favor of the penetrating cryoprotectants 3-methoxy-1,2-propanediol and n-methyl-formamide, and increased concentration of the ice-blocker Z-1000. The final molar concentration of 9.345 M demonstrates that more concentrated vitrification agents do not necessarily have to be more toxic.





Dimethyl sulfoxide

3.10 M

3.10 M

2.855 M

2.855 M


3.10 M

3.10 M

2.855 M

2.855 M

Propylene glycol

2.21 M

Ethylene glycol

2.71 M

2.713 M

2.713 M


0.508 M


0.377 M

Polyvinyl pyrrolidone K12*

7% w/v

2.8% w/v

X-1000 ice blocker*

1% w/v

1% w/v

Z-1000 ice blocker*

1% w/v

2% w/v

Total Molarity

8.41 M

8.91 M

8.41 M

9.345 M

* Non-penetrating polymers are in w/v

M22, so called because it was intended to introduced at -22 degrees Celsius, constitutes a major landmark in vitrification of complex organs. In 2005 Fahy, Wowk et al. announced routine recovery of rabbit kidney slices from temperatures around -45 degrees Celsius. Although consistent recovery of vitrified organs is not yet feasible, continued progress in solution composition and perfusion techniques inspire optimism that this may be possible in the future. In 2007, Greg Fahy of 21CM reported recovery of electrical activity in vitrified brain slices and induction of long-term potentiation (LTP), which indicates that the structures for processing memory are maintained after vitrification, storage and rewarming of brain tissue. Visual evidence that M22 can preserve the ultrastructure of the brain better than B2C was published on the Alcor website in 2005.

M22 also needs to be used in a suitable carrier solution to support cell metabolism at low temperatures and decrease oxidative injury and edema. The carrier solution for M22 is called LM5 to reflect the 50% reduction of glucose (as compared to the older carrier solution RPS-2) in favor of equimolar concentrations of mannitol and lactose, to address compatibility problems with the ice blockers. The combination of the isotonic LM5 plus the non-penetrating polymers in M22 creates a hypertonic solution, which has been shown to eliminate chilling injury, which is the injury that is caused by exposure to low temperatures as such. For cryonics, the composition of M22 is further enhanced by including a proprietary components that allows perfusion of whole body patients without edema.

The research breakthroughs discussed above allow for a global reconstruction of the composition of M22 using the table. Maintained is the equimolar combination of DMSO and formamide from Fahy’s older vitrification solutions to reconcile strong glass formation ability and minimal toxicity. The discovery of the  compositional variable qv* allows for substitution of higher concentrations of the weaker glass former ethylene glycol for propylene glycol. Substitution of a non-penetrating polymer, PVP K12, and the ice-blockers X-1000 and Z-100 allow for further reduction of DMSO and formamide, reduction of critical cooling rates, and increased stability against ice formation. In M22, PVP K12 is reduced to optimize hypertonicity of the non-penetrating agents for suppression of chilling injury. Added are the methoxylated cryoprotectant 3-methoxy-1,2-propanediol and the highly permeable amide n-methyl-formamide, producing the least toxic but most concentrated vitrification solution to date.

The most striking differences between Alcor’s old perfusate and the newer vitrification agents licensed from 21CM are complexity and cost. Until 2002, Alcor patients were perfused with high molar glycerol in an MHP-2 based carrier solution. M22 itself consists of 8 (!) different components, putting the total number of components of M22 in carrier solution above 15. Such perfusates makes great demands on preparation skills and quality controls. Components such as the ice blockers and 3-methoxy-1,2-propanediol have put the cost of Alcor’s whole body perfusate alone close to the cost of complete cryopreservation arrangements at the Cryonics Institute (CI). This raises obvious questions about costs and benefits. As evidenced by CI’s VM-1, potent protection against ice formation can be achieved with a vitrification agent that solely consists of DMSO and ethylene glycol. It is plausible to assume that vitrification lessens demand on future repair technologies, but it speculative to assume that minor differences in toxicity between different vitrification agents will translate in earlier resuscitation and less expensive repair protocols. However, more toxic vitrification solutions, such as CI’s VM-1, may cause acute injury to endothelial cells. As Brian Wowk notes, “good cryoprotection depends on good perfusion, which depends on preservation of vascular integrity during perfusion. The ability to perfuse M22 into whole bodies with tolerable edema is likely to be intimately related to its low toxicity to vascular endothelium.” And of course, there are also PR advantages to the fact that a cryonics organization uses a vitrification agent that is also the state of the art in conventional cryopreservation of organs.

M22 produces substantial brain shrinking during perfusion of (non-ischemic) patients. As a matter of fact, cerebral dehydration may be a major contributing factor to vitrification of the brain and even allow for reduced concentrations of M22 for brain preservation. This does not mean that the (expensive) non-penetrating polymers could be replaced for any high molecular weight polymer because the ice blockers and non-penetrating cryoprotective agents also protect the extracellular space against ice formation and are effective in ischemic patients with a compromised blood brain barrier (BBB). The limited ability of some components of M22 to cross the BBB and, and differences in permeability of the various components of M22, does raise questions about the exact composition of M22 beyond the BBB and within brain cells after completion of cryoprotective perfusion.

Patients outside of the US may not fully benefit from cryopreservation with M22 because of the of long cold ischemic times during transport. This raises the question if cryonics patients can be perfused outside of the US and shipped in dry ice. Experiments with VM-1 in bulk solution indicate that this solution is very stable against ice formation, even during long storage periods. M22 in bulk solution seems to form ice crystals overnight if stored in dry ice. This does not necessarily mean that M22 cannot be used in combination with dry ice for overseas patients because human tissue perfused with M22 (or any cryoprotective agent) is not the same as M22 in pure solution. But regardless of M22’s compatibility with dry ice shipping, cryonics organizations may benefit from formulating a highly concentrated inexpensive vitrification solution that is extremely robust against formation of ice, which can be used for simple perfusion of non-US patients in combination with dry ice shipping. The decreased cold ischemic times of such a solution may outweigh the increased toxicity of such solutions.

Cerebral blood flow during and after cardiac arrest

As discussed in a previous post, perfusion of the brain following long-term (>5 min) ischemia has been shown to be significantly compromised, particularly in subcortical regions. An interesting recent article by Ristagno, et. al in Resuscitation (May 2008) has added new data to the equation, using some of the most advanced technologies available for measuring cerebral microvascular blood flow.

To briefly summarize the experiment, pigs were subjected to 3 minutes of untreated ventricular fibrillation followed by 4 minutes of cardiopulmonary resuscitation and subsequent defibrillation. Blood flow in large (>20 micrometers) and small (<20 micrometers) cerebral vessels was measured during and after CPR by direct visualization using orthogonal polarization spectral imaging (OPS) together with cortical-tissue partial pressure of carbon dioxide.

Though prior studies implied a dissociation between microcirculatory flow and macrocirculation during CPR, Ristagno, et. al found “a close relationship between microvascular flows and the macrocirculation during cardiac arrest, during CPR and following return of spontaneous circulation (ROSC).” Interestingly, they also noted that cerebral blood flow was reduced, but did not stop, for more than 2 minutes after cardiac arrest, most likely due to residual compliance in the arterial circuit. After ROSC, flow progressively increased back to normal (pre-arrest) values within 3 minutes.

Importantly, the researchers also noted that cerebral cortical-tissue partial pressure of carbon dioxide (a measure of the severity of cerebral ischemia) increased progressively througout CPR, providing evidence for the fact that the pressure and flow generated during chest compressions “may minimise but do not reverse the magnitude of the brain ischaemia which preceded the start of CPR.”

Though many investigations, such as the previously reported study by Fischer & Ames reported no-reflow or hypoperfusion following ischemia, these authors observed no such phenomena, possibly because of the short duration of cardiac arrest. Indeed, they ultimately conclude that “a 3-min interval of ischaemia was therefore probably not long enough to induce alterations in blood flow during reperfusion.” Also of importance is the fact that OPS technology limits visualization of microvessels to within 1mm of the cortical surface. However, this paper still gives us better insight into the immediate effects of cardiac arrest, cardiopulmonary resuscitation, and reperfusion on microcirculatory flow in the brain.

Albert Einstein's brain and information-theoretic death

People like you and I, though mortal of course like everyone else, do not grow old no matter how long we live…[We] never cease to stand like curious children before the great mystery into which we were born.”

Albert Einstein

One sign of the lack of faith in the future progress of technology and the poor acceptance of the neurological basis for mind is the way in which our society treats the “post-mortem” human brain.

In some cases, the brains of those whom modern medicine cannot help are removed after cardiopulmonary arrest and donated (by the permission of the patient or the family) for research. In such cases, the brains are preserved so they can be studied over a long period of time. They are also sectioned and prepared in other ways for examination. Such donated brains have helped scientists learn about the human brain, with an eye to improving methods for treating conditions such as Alzheimer’s or mental illness. However, other brains have been preserved mainly because they belonged to famous people.

One of the more famous cases is the brain of Albert Einstein, removed in 1955 and preserved apparently without his or his family’s permission, and then made available for study. According to an NPR report, Einstein’s brain was fixed, sectioned into over 200 blocks, embedded in celloidin, and then stored in formalin.

Since that time, Einstein’s brain has been further sectioned and divided among researchers. A 1985 study by Diamond et al. reported that the Einstein brain sections’ neurons were still observable, and the study’s authors even assumed the number of neurons preserved in Einstein’s brain would be the same as those in recent preserved brains.

Presumably, people have wanted to study the brains of famous people in order to learn something about what made those people special. Turning a person into a mere object of study is a questionable notion, though, and the idea that the study could yield any information about the person’s mind underscores how it is widely accepted by scientists that the brain instantiates the mind, and thus the person.

Neuroscience is still too much in its infancy to make much sense of the evidence of the brain, as the scientific reception to the Diamond study showed. We do not yet know how to “read” the brain for the specific memories and personality traits and other phenomena of mind stored in it. However, because we do know enough now to know that the mind arises from the brain, we must realize that to preserve the brain is to preserve the potential of mind, and to preserve the potential of mind is to preserve the possibility of life for the person whose brain it was.

The neural basis of personhood sits ill with older notions such as immaterial souls or spirits. The neural basis of personhood also fits poorly with existing medical and public policies such as commonly accepted definitions of death and laws related to end of life. If death is understood as irreversible damage to certain identity-critical areas of the brain, the irreversibility of such damage is put into question by every advance in the treatment of injury and disease of the brain, as well as by the brain’s mysterious ability to recover from conditions such as minimally conscious state after many years. The cardiopulmonary-arrest definition of death does not involve the condition of the brain, and the usual definitions of brain-death do not distinguish between identity-critical areas or aspects of the brain and other areas or aspects of the brain. A more rigorous definition of personal death has been developed by Ralph Merkle, who states:

“A person is dead according to the information-theoretic criterion if their memories, personality, hopes, dreams, etc. have been destroyed in the information-theoretic sense. That is, if the structures in the brain that encode memory and personality have been so disrupted that it is no longer possible in principle to restore them to an appropriate functional state then the person is dead. If the structures that encode memory and personality are sufficiently intact that inference of the memory and personality are feasible in principle, and therefore restoration to an appropriate functional state is likewise feasible in principle, then the person is not dead.”

Although there is still some lack of clarity about the “etc.” and “appropriate functional state”, this definition of death at least is founded on the neural basis of personhood. Those who believe in the future progress of technology and accept the neural basis of personhood are led inevitably to understand that preserving the brain is preserving the person, potentially for later resuscitation.

It is not impossible to imagine that, in a more advanced future time, the formalin-fixed, celloidin-embedded brain sections could be reassambled, and if the synaptic circuitry of the neurons were well preserved, any significant damage could be repaired. The brain might be able to be returned to a viable state by reversal of the fixation and removal of the celloidin embedding. Resuscitation of an isolated brain would be unacceptable, but eventually it might be possible to restore the rest of the body around the brain by cloning or regeneration of the cells or some other prosthetic embodiment.

As amazing as it may seem, a patient reduced to a preserved brain, whose mind would be in a stopped state, might be able to be healed, that is, totally restored to a healthy body and a mind which could resume the life it left off, with all the memories and personality intact.

The case of Albert Einstein’s brain is unfortunate. All the impudent cutting, handing around, and tampering with Einstein’s brains sections, and the crude preservation method, may have irreversibly damaged the neural basis of his personhood. Yet we do not know enough today about the brain to know how much of it needs to be preserved, and in what state, to be able to revive a person with future technology. The preservation of the brain, though, would provide a theoretical possibility of future resuscitation. It may not be possible to someday restore Albert Einstein from the remains of his brain, but if it were possible, those in possession of the brain sections would first have to be willing to consider whether their “specimens” might be the restorable fragments of a still potentially living person who deserves to live more than to be studied.

Fever and brain injury

Elevation of body temperature occurring as a result of hypothalamic coordination of autonomic, neuroendocrine, and behavioral responses in reaction to physiological injury or invasion is generally known as fever. Traditional thought is that the “febrile response” is beneficial in preventing the proliferation of invading microorganisms, but some caregivers consider fever to be harmful and prescribe antipyretic agents and/or physical cooling methods to suppress fever. In their recent publication, Aiyagari and Diringer summarize the data that exists concerning the efficacy of physical and pharmacological treatments in reducing temperature and improving outcome in a variety of acute neurological disorders including stroke, traumatic brain injury, and cardiac arrest.

Several rationales exist for treating fever, including the relief of discomfort associated with fever, reduction of fever-imposed increase in metabolic demand, reduction in morbidity and mortality, reduction of fever-induced cognitive impairment, and prevention of febrile seizures. Most of these rationales are beneficial in theory, but have not been proven in practice. In the case of morbidity/mortality reduction, treatment with antipyretics has been shown to prolong certain infections; similarly, fever is known to improve survival of patients with community acquired pneumonia, Eschericia coli bacteremia, and Pseudomonas aeruginosa sepsis. Compounding these issues is the fact that traditional methods of lowering temperature in febrile patients are ineffective.

Elevated temperature exacerbates neuronal injury caused by cerebral ischemia or traumatic brain injury (TBI) and, conversely, hypothermia acts as a neuroprotectant in such cases. Well-controlled animal models of global and focal ischemia demonstrate a significantly detrimental effect of hyperthermia on clinical outcome and neuropathological changes. Ginsberg and Busto ( 1998 ) list a number of mechanisms through which hyperthermia worsens outcome in cerebral ischemia: increased neurotransmitter release, increased free radical production, opening of the blood-brain barrier, increased depolarizations within the penumbra, impaired brain metabolism and second messenger inhibition, and cytoskeletal degradation. The authors also note that “the action of otherwise neuroprotective drugs in ischemia may be nullified by mild hyperthermia.” Meticulous brain temperature monitoring and treatment of elevated temperature in patients suffering from neurological insult may, therefore, help prevent secondary injury.

Clinical studies of TBI and survivors of cardiac arrest have demonstrated an independent relationship between fever and poor outcome. Although fever is extremely common in neurological intensive care unit patients, the lack of effective fever treatment options has severely limited the availability of data regarding the benefits of fever reduction in such patients. However, recent advances in surface and intravascular cooling devices have lead to improvements in ability to reduce temperature, especially in patients with neurological injuries. An external cooling device known as the Medivance Arctic Sun temperature management system appears to be quite effective at reducing temperature in febrile patients (75% reduction in fever burden) as compared with more traditional means of fever reduction such as air- or water-circulating cooling blankets. Similarly, a newly-devised catheter-based heat exchange system (Cool Line/Cool Gard) has been tested in patients with subarachnoid hemorrhage (SAH), intracerebral hemorrhage (ICH), cerebral infarction, or TBI, showing a 64% reduction in fever burden as compared to the conventional treatment group (antipyretic, cooling blanket, and ice packs). Unfortunately, no data exists concerning these interventions’ impact on outcome.

As cooling devices and methods are improved and proven to be effective, more data concerning the effect of fever reduction on outcome should be forthcoming. Importantly, as Aiyagari and Diringer point out in the conclusion of their review:

“In the absence of conclusive data, the approach to fever management should be based on the balance between the potential for fever to exacerbate brain insults vs. enhance the ability to fight infections. Fortunately, the risk of ongoing brain injury is usually limited to the early phase in the course of most acute insults while the risk of infection rises as time goes on. Thus it would seem reasonable to aggressively control fever during the first few hours to days following ischemic stroke, intracerebral hemorrhage and head injury. Subsequently, aggressive fever control is less likely to be of help and could be detrimental.”

In cryonics patients, infection exacerbation is less important than protecting the brain from injury and warrants immediate induction of rapid cooling to protect patients from injury due to elevated temperatures. The benefits of treating fever in brain injury also highlights the importance of maintaining normothermia, or even hypothermia, in agonal (hypoxic) patients that present for cryopreservation.

In situ chemical fixation of whale brains

As discussed by R. Michael Perry in his recent contribution to Cryonics Magazine, “Alternatives to Cryonics: A Very Preliminary Study,” (3rd Quarter 2007) chemical fixation of the brain may be a substitute for cryopreservation in circumstances where cryonics is not feasible or affordable. Several issues come into play when attempting to determine whether chemical fixation results in acceptable preservation of ultrastructure. An important question is whether chemical fixatives are uniformly distributed throughout the brain, preventing the occurrence of islands of tissue decomposition due to inadequate fixation.

In their 2002 paper, Knudsen et al. provide some preliminary answers to this question. Due to the difficulty in obtaining fresh brains for study from large aquatic mammals, the researchers developed a novel method for in situ fixation of (minke) whale brains. The procedure involved cutting a triangular opening in skull and pouring 8% formalin solution into the epidural and subdural space until it leaked out through the foramen magnum. The foramen magnum was then plugged, the triangular bone piece replaced to reduce loss of fixative, and the fixative level was checked and refilled every 4 hours, if necessary. Pilot studies indicated that a diffusion period of at least 60 hours is required for adequate fixation of large volume (average = 2201 g) minke whale brains, so the researchers set the in situ fixation period at 72 hours “to ensure that even the largest brains were sufficiently fixed prior to excision.” The brains were then excised and stored in formalin for at least 2 months prior to gross and microscopic examination.

Gross examination revealed that “there were no cases where the brain tissue was liquefied or smelled sour due to post mortem bacterial growth and the occurrence of artifacts and autolytic changes due to incomplete fixation was generally low.” Microscopic examination showed well-preserved cells and myelin in all parts of the brain. Specifically, histological evaluation categorized 97.3 to 100% of samples taken from different sites (brain stem and spinal cord, cerebellum, and cerebrum) as ‘good’ fixation (occurrence of mild autolytic changes) or ‘excellent’ fixation (without autolytic changes). “Swiss cheese” artifacts, caused by the invasion of gas-forming anaerobic bacteria, were observed in restricted (central) parts of the brain in 22 of 38 brains, especially in the thalamus, brain stem, and cerebellar vermis. White matter vacuolation was also observed in some of the brains, again in the thalamus and cerebellar vermis. However, in every case, the vacuoles were few (1 to 5) and small (1 to 3 mm).

The authors conclude that “the subsequent histological examination showed that these brains were, in many ways, better preserved than the routine autopsy brains of human and veterinary medicine. We regard the time span from death to start of fixation as the most decisive or crucial factor for this successful result.” They indicate that, although ≈75% of the fixations began within 2 hours post mortem, there were some instances where fixation started later (up to 6 hours), and that variations in fixation quality are likely due to the occurrence of autolytic changes. Importantly, it was noted that even careful handling of fresh brains always results in compression damage, and that fixation of the brain in situ was an excellent remedy for this problem.

In situ whale brain diffusion fixation appears to produce good preservation of the structure of the whole brain, especially in cases where fixation is begun soon after death. If such results can be achieved by passive diffusion of vastly larger brains than the human brain, investigation of the feasibility of reproducable uniform chemical fixation of complete humans brains as a method of biopreservation is warranted.

Characterization of afterhyperpolarization (AHP) in cryopreserved brain slices

An ongoing quest in cryonics is the successful demonstration of memory sustainment after cryopreservation of the brain and rewarming from cryogenic temperatures. To that end, landmark experiments were performed by Pichugin, et al. (2006) on rat hippocampal brain slices which indicate that the hippocampus retains excellent structural integrity and viability (as measured by Na+/K+ ion pump recovery) after vitrification, rewarming, cryoprotectant removal, and exposure to 35°C for over an hour. To address the question of memory itself, investigations into the maintenance of long-term potentiation (LTP) after vitrification of the brain are currently in progress. But even successful observation of LTP after cryopreservation provides only indirect evidence for memory maintenance.

Alternatively, post-burst afterhyperpolarization (AHP) of hippocampal CA1 neurons may be characterized after cryopreservation of animals that have successfully acquired a hippocampus-dependent task. CA1 pyramidal neurons show decreased post-burst AHPs and less accommodation (i.e., increased firing frequency) following learning of such hippocampus-dependent tasks as trace eyeblink conditioning (Moyer et al., 1996, 2000; Thompson et al., 1996) and spatial watermaze training (Oh et al., 2003) with a time course appropriate to support memory consolidation. Furthermore, CA1 neurons of aging animals (i.e., animals at ages that exhibit learning deficits) show greater AHPs and more accommodation than those of young animals (Landfield & Pitler, 1984; Moyer et al., 1992, 2000), indicating an age-related decrease in neuronal excitability in the hippocampus that may underlie learning deficits related to aging.

A carefully designed experiment demonstrating reduced afterhyperpolarization and accommodation in hippocampal CA1 neurons after acquisition of a hippocampus-dependent task and subsequent cryopreservation of the brain would be a huge step in the direction of proving that memories can be cryopreserved.

Whole body cryopreservation with preferential brain treatment

A strong argument in favor of neuropreservation is that all efforts can be devoted to vitrification of the brain. Perfusion times are shorter and challenges present during perfusion of the rest of the body (such as abdominal swelling and the higher viscosity of whole body perfusates) are eliminated. The technique of isolated head perfusion may offer additional advantages such as increased cooling rates, superior venous drainage and reduced facial edema.

Some of these advantages are not incompatible with whole body cryopreservation if preferential treatment of the brain is offered to whole body patients. A number of recent Cryonics Institute cases, such as CI-77, indicate that such an approach may be feasible if experimental and practical challenges involving the composition of the cryoprotectant agent, cannulation and perfusion techniques, gastrointestinal ischemia, and selective brain cooling are overcome.

Future installments of this blog will review these separate issues in more detail.