by Aschwin de Wolf
The main objective that guides care in cryonics is to maintain viability and preserve the ultrastructure of the brain during all procedures. Because of its high metabolic demand and low capacity for energy storage, the brain is extremely vulnerable to injury caused by lack of blood flow (cerebral ischemia). The ability to secure viability and good ultrastructural preservation of the brain is therefore an excellent measure of the current state of the art in cryonics. Because identity and memory are assumed to reside primarily in the brain, both whole body and neuropreservation members would agree that this organ should be given preferential treatment. This article will briefly describe all the steps involved in a typical cryopreservation case and discuss how far we have come in achieving this objective in cryonics
Structure versus Viability
One distinction that is often made in cryonics is that between ultrastructure and viability. In this context viability means that the brain is able to resume function upon reversal of some, or all, of the procedures employed in cryonics. Preservation of ultrastructure refers to preservation of the detailed structure of a cell, tissue, or organ that can be observed by electron microscopy. Naturally, these two concepts are related. For example, if an organ were straight frozen (placed into liquid nitrogen without cryoprotectant perfusion) after a long period of warm ischemia, we would expect to find poor ultrastructure and, therefore, poor viability. But there can also be examples where good preservation of ultrastructure does not necessarily guarantee a good outcome in terms of viability. Examples of this would be procedures that result in good preservation of ultrastructure but which cause mitochondrial failure, denatured proteins, or massive activation of apoptosis (programmed cell death).
One aspect often neglected by cryonics writers is that many patients who present for cryonics go through a prolonged terminal period before cryonics stabilization procedures are initiated. During this period the patient may experience a number of pathological conditions such as shock, respiratory distress, dehydration, electrolyte imbalances, systematic inflammation, upregulation of coagulation factors, multiple organ failure, intracranial pressure, and activation of apoptosis. Consequently, the objective of stabilization, is much more difficult to achieve or may even be defeated before the cryonics team gains access to the patient.
Because cryonics organizations do not treat the patient before legal pronouncement of death, it is largely the patient’s responsibility to execute the proper paperwork to ensure that medical treatment during the terminal period will not be detrimental to achieving a good cryopreservation. Examples that come to mind are to have a Do Not Resuscitate (DNR) order in place to avoid multiple resuscitation attempts (with associated cycles of ischemia-reperfusion injury) and to express a desire for certain supplements during palliative care. Where the cryonics organization can make a difference during this period is in being guided by the “premortem” condition of the patient when starting stabilization procedures such as promptly restoring fluid volume and vascular tone after a patient has been pronounced dead.
Cryonics stabilization procedures consist of three different interventions: cardiopulmonary support (CPS), induction of hypothermia, multi-modal medications treatment, and in remote cases, blood washout and substitution with an organ preservation solution. Stabilization of the patient is one part of cryonics protocol where a number of cryonics authors have explicitly stated that cerebral viability by contemporary medical criteria should be the objective . In this vein, Alcor and associated research companies have done research to demonstrate that securing cerebral viability during stabilization is a realistic objective.
Two groundbreaking experiments provide evidence that securing viability during stabilization might be achieved with current technologies. In the late 80s and early 90s Darwin, Leaf et al. demonstrated that induction of ultra-profound hypothermia (temperatures lower than 5°C) in conjunction with blood washout and substitution with an organ preservation solution is reversible in a canine model. Dogs were revived from up to 5 hours of low flow perfusion with an organ preservation solution called MHP-2 . In the mid 90s Darwin, Harris et al. successfully resuscitated dogs from up to 17 minutes of normothermic cardiac arrest using a large number of medications and tight post-resuscitation regulation of hemodynamics .
Impressive as these results are, a number of caveats need to be taken into account. First, as mentioned previously, the typical patient who presents for cryonics has gone through a prolonged terminal period. How realistic is it to expect a similar outcome under such conditions? Second, current cryonics procedures are not identical to the protocol that was investigated during these experiments. For example, in remote cases the organ preservation solution is used in a static (no flow) fashion instead of constantly perfusing the patient at low flow during transport to the cryonics facility. In the case of the normothermic cerebral resuscitation experiments it is also important to note that the dogs were pre-heparinized prior to cardiac arrest (heparin is administered after cardiac arrest in cryonics cases) and that resuscitation doesn’t involve a long period of external chest compressions as is the case in cryonics stabilization. Finally, some techniques that are possible during experimental work in a laboratory – such as rigorous medications administration, tight control over hemodynamics and sophisticated monitoring – are currently not available to cryonics
The objective of securing viability during cryoprotective perfusion can be broken down into two stages. During the initial phase, following surgery to obtain vascular access, the patient’s blood (or the organ preservation solution in a remote case) is flushed out. Because this phase is not fundamentally different from remote blood washout, securing cerebral viability should be possible in principle, provided that the transition from stabilization to initiation of OR procedures is structured in such a fashion that that there is (1) no major interruption in circulation or (2) no marked rise in temperature. Unlike the first condition, the latter condition is not only a practical challenge but a clinical challenge as well. Effective washout is a function of temperature and this presents a delicate trade-off between the risk of ischemic injury produced by elevated temperatures and the benefit of reduced washout times.
A related problem is encountered in the second phase of perfusion during which a cryoprotective agent is gradually introduced to the patient. Classical cryoprotective agents like glycerol do not penetrate cell membranes very well at lower temperatures (close to 0°C). To compensate for this fact, in the past sometimes a deliberate elevation of temperature was required during glycerol-based cryoprotection. Although this was a rational choice (considering the alternative of extremely long perfusion times), the introduction of a very concentrated cryoprotective agent at relatively high temperatures likely compromised cerebral viability as a result of increased ischemic exposure and cryoprotectant toxicity. Current cryoprotective agents are no longer based on glycerol and include components such as DMSO, which have improved permeability at lower temperatures.
The real limiting factor for maintaining viability of the brain is that all currently known cryoprotectants have toxic effects when whole brains are exposed to them long enough to prevent ice formation and achieve vitrification during cooling. At the time of writing, the M22 cryoprotectant mixture used by Alcor is the least toxic vitrification solution ever published for use in large organs . However, it is still not sufficiently non-toxic to permit reversible cryopreservation of the whole brain. Another reason why cerebral viability might be compromised during introduction of cryoprotectants is that, under “ideal” circumstances, the vitrification agent induces an extreme degree of brain shrinking which may compromise vascular and cellular integrity and even set the stage for apoptosis upon resuscitation. Overcoming these problems will require further advances in basic research.
Cryogenic Cooldown and Long- Term Care
Because we can deduce that cerebral viability is lost during the later stages of cryoprotective perfusion, we know that cerebral viability can no longer be maintained during cryogenic cooldown and long-term care of the patient. In general, if cerebral viability is lost at some earlier phase, it cannot be restored during any later phase of cryonics procedures. Consequently, the emphasis from that point will be on preserving ultrastructure as best as possible. During cryogenic cooldown this means cooling at least fast enough to inhibit any ice formation, which is currently 0.1°C/minute for the cryoprotectant M22. A cooling rate of ~ 0.4°C/ minute can be achieved for an organ as large as the human brain. Since an adequate cooling rate can be achieved to prevent ice formation in the brain, the remaining issues of immediate concern include cryopreservation-induced injuries independent from ice formation like chilling injury and thermal stress at lower temperatures.
Chilling injury involves injury caused by exposure to low temperatures as such and includes cell membrane phase transitions and protein denaturation. Although M22 was designed to prevent chilling injury in large organs, this problem has not been investigated in cryonics patients. Aside from the practical problems in identifying chilling injury during cryoprotective perfusion and cooldown, it may be hard to distinguish the effects of chilling injury from the injury caused by warm ischemia, cryoprotectant toxicity, and osmotic shock. Moreover, chilling injury may be relatively benign compared to other problems during cryopreservation, such as the risk of ice formation and thermal stress.
Below the glass transition temperature (-123.3°C for M22) the vitrification solution turns into a glass and is limited in its ability to further contract as the temperature is further lowered, causing tissues to fracture as a result. Thermal stress not only presents an obvious obstacle to maintaining viability, but fracturing also compromises the objective of securing uniform ultrastructure of the brain. In light of the expectation that contemporary vitrification solutions will inhibit ice formation in cryonics patients, eliminating fracturing has become a more urgent priority in cryonics. One solution would be to provide long-term care for patients at higher temperatures, just below the glass transition point. Another alternative would be to develop an “annealing” protocol that will inhibit, or minimize, thermal stress by keeping a firm control over temperature descent .
Leaving social, political, and legal threats to cryonics patients aside, the final challenge to securing cerebral viability for cryonics patients is the effect of long-term care on the patient. Although viability and ultrastructure have already been compromised by current procedures at this point, there is no reason to believe that long-term care at liquid nitrogen temperature (-196°C) would produce adverse effects over very long periods of time (exceeding thousands of years) . At the temperature that cryonics patients are currently maintained, time has effectively been halted. Open to more debate is the long-term risk of maintaining patients at intermediate temperatures (slightly under the glass transition temperature) because, at temperatures down to 20°C below the glass transition temperature, ice nucleation may still be a risk for cryopreservation. These nanoscale nucleators may not present a direct threat to patients during long-term care, but they may present a bigger challenge during rewarming of the patient in the future.
How do we know if cerebral viability is being maintained during a cryonics case? At this point most evidence for what is possible with current cryonics technologies has come from experiments on healthy animals under controlled laboratory conditions. In light of the fact that cryonics procedures do not occur under such tightly controlled circumstances, claims that viability can be secured up until the later stages of cryoprotective perfusion must remain highly theoretical.
Currently the only means available to cryonics organizations to get an idea about how well cerebral viability is being maintained during stabilization and cryoprotective perfusion are confined to physiological observation, temperature data, qualitative end tidal CO2 and peripheral oxygen saturation readings and, in rare cases, pre-pronouncement and post-pronouncement blood gases and electrolytes. For example, a case where cerebral viability is maintained would typically have all, or most, of the following characteristics: prompt start of stabilization procedures after legal pronouncement of death, adequate cerebral perfusion generated by mechanical chest compression (or extracorporeal perfusion) and administration of vasoactive medications, and rapid induction of hypothermia.
During a number of landmark cases at Alcor and CryoCare blood gases and temperature data have been collected that seem to indicate that viability may have been maintained during stabilization . However, when reading these case reports it should be kept in mind that more subtle ischemic changes may have occurred that still present a threat to viability such as mitochondrial damage, excessive free radical damage, activation of apoptosis, or neurological pathologies associated with induction of ultra-profound hypothermia and extracorporeal perfusion. Consequently, blood plasma should be examined to look for more specific biomarkers of brain injury.
From initiation of cryogenic cooldown to long-term patient care, measurements of viability are no longer possible and cryonics organizations confine themselves to optimizing preservation of ultrastructure. During cooldown Alcor uses an acoustic monitoring device to monitor the presence of fracturing in the brain. This device uses an electronic sensor that registers vibrations that are assumed to correspond with fracturing events. After cryogenic cooldown the only available method to determine whether any ice has formed is direct observation of the surface of the brain. Naturally, during long-term care at liquid nitrogen temperatures neither measurements of viability or ultrastructure can be taken in real time.
One may wonder why some cryonics organizations make such an effort to maintain cerebral viability during stabilization if it is invariably lost during cryoprotective perfusion and cryogenic cooldown. The straightforward answer is that by securing viability at an early stage, better preservation of ultrastructure can be achieved at a later stage. Cardiac arrest sets the stage for a number of pathophysiological events that can interfere with optimal circulation of the cryoprotective solution during the later stages of cryonics procedures including, but not limited to, intravascular blood clotting, production of inflammatory vascular adhesion molecules, free radical formation and capillary and cell membrane leakage. Notable differences in cryoprotective perfusion have been observed between patients that experienced a long period of warm and/or cold ischemia and patients who received prompt stabilization and minimal transport times.
A related but more subtle issue is whether cryonics stabilization protocol could benefit by changing the objective of stabilization from securing cerebral viability to optimizing cryoprotective perfusion. Typically one would expect that interventions that are adequate to secure viability will also confer benefits during cryoprotective perfusion, but there at least three caveats to this perspective that need to be considered.
First, there are interventions that can secure viability if executed promptly and correctly but that can frustrate cryoprotective perfusion at a later stage in the absence of a careful approach. Ventilating a patient with 100% oxygen is an example of an intervention that might be moderately beneficial in terms of viability but can also seriously frustrate adequate distribution of the cryoprotective agent in the brain as a result of injury to the circulatory system and cell membranes (a condition known as “reperfusion injury”). Second, there are only a finite number of pharmacological interventions that a cryonics organization can be expected to do and a choice needs to be made between interventions that increase the probability of short-term recovery and a protocol that is specifically designed to preserve ultrastructure through all phases of cryonics procedures. This choice is especially important in light of the fact that Alcor’s medications protocol reflects a normothermic recovery model to mitigate a number of pathophysiological events that should also be inhibited by rapid induction of hypothermia. Third, Alcor’s organ preservation solution, MHP-2, has never been investigated for prolonged static use or in the presence of serious ischemic and reperfusion injury. In general, results obtained in a recovery model need to be validated in a model that reflects the typical patient pathologies and practical limitations of a cryonics standby team.
Should viability of the brain be the golden standard for cryonics care anyway? We can imagine a scenario where a cryonics patient can be successfully resuscitated but with impaired personality and memories. For example, it is a well established fact that the CA1 region of the hippocampus in the brain is highly vulnerable to even the shortest interruptions of cerebral blood flow. This region of the brain is associated with encoding and storing memories. Cryonics would benefit from a better understanding why certain regions of the brain are so vulnerable to oxygen deprivation to guide research into procedures that minimize injury to vulnerable cells in the brain. Getting a better understanding of the efficacy of current procedures, and improving upon them, is one of the objectives for reviving the ambitious research agenda that cryonics pioneers Jerry Leaf and Mike Darwin pursued at Alcor. Current cryonics organizations are also investigating a number of technologies that will improve cardiopulmonary support, rapid induction of hypothermia, optimize control and data collection during cryoprotective perfusion, and reduce fracturing during cryogenic cooldown.
Despite the renewed focus on evidence based cryonics and new technologies, one of the major limiting factors in securing viability and good ultrastructure is the quality of standby and stabilization procedures. This objective requires a concerted effort among cryonics organizations and their members ranging from forming new local cryonics groups to making substantial investments to distribute good stabilization equipment in many parts of the country.
An earlier version of this article was published in Cryonics Magazine, 2nd Quarter, 2007
1. Wowk Brian, “Cardiopulmonary Support in Cryonics”, Alcor Life Extension Foundation.
2. Leaf Jerry D, Darwin Michael G, and Hixon Hugh, “A mannitol-based perfusate for reversible 5-hour asanguineous ultraprofound hypothermia in canines.” Cryovita Laboratories, Inc. and Alcor Life Extension Foundation, unpublished paper 1987.
3. This work has been done by Critical Care Research. Unpublished research results.
4. “New Preservation Technology”, Alcor Life Extension Foundation.
5. “Cryopreservation and Fracturing”, Alcor Life Extension Foundation.
6. Hixon Hugh, “How Cold is Cold Enough”, Cryonics Magazine, January 1985.
[Note that this article presents a rather optimistic source for the time that we can expect a human to be resuscitated from cardiac arrest.]
7. Darwin Michael G, “Cryopreservation Patient Case Report: Arlene Frances Fried, A-1049”, Alcor Life Extension Foundation and Darwin Michael G, “Cryopreservation of CryoCare Patient #C-2150”, Biopreservation Tech Briefs (1996, no. 18)