From Cryonics, September 2013
by Chana Phaedra
As all cryonicists understand, the one absolutely necessary component of the human body that must be preserved in order for one to be successfully resuscitated from cryonics is the brain. The last major review of the state of brain cryopreservation can be found in “The Cryobiological Case for Cryonics,” published in the March 1988 Cryonics. This article will very briefly touch on that report (please, read the entire piece on the Alcor website) then supplement it with progress in the field since then.
One of the most important points that “The Cryobiological Case for Cryonics” makes is that because biological systems are composed of different cell types “it is difficult to extrapolate from one biological system to another in terms of predicting the details of its cryobiological behavior.” And because traditional cryobiologists have largely ignored the brain, we must endeavor to seek out information and run experiments that focus specifically on the brain.
Fortunately, the results of such investigations have been promising. In 1988 we were already able to state that “wherever either brain structure or brain function has been evaluated after freezing to low temperatures and thawing, robust preservation has almost always been demonstrable provided at least some minimal attention was paid to providing cryoprotection…” This is remarkable, and is backed up by numerous examples ranging from the landmark hamster-freezing experiments of Lovelock and Smith in 1956 to the cat-brain EEG studies of Suda and colleagues in the 1970s, both demonstrating a return to function in living adult animal brains after freezing.
Lovelock and Smith used no cryoprotectants in their experiments, freezing and then resuscitating hamsters from high subzero temperatures of around 3 to 5°C. Suda et al., on the other hand, used low concentrations of glycerol (~15%) to cryoprotect cat brains before freezing at 20°C, 60°C, or -90°C and storing them for periods ranging from 5 days to 7.25 years. Amazingly, Suda reported recovery of single-unit and EEG activity in these brains. In general, neural function was better when brains were stored at higher temperatures for short periods of time, and worsened when stored at lower temperatures for longer periods of time.
Later on in the 1980s, Fahy and colleagues reported excellent histological preservation of the cerebral cortex and hippocampus after cryoprotection with 3M or 6M glycerol and slow freezing to dry ice temperature (79°C), demonstrating that cryoprotection and freezing is capable of preserving the cellular structure of the brain as well.
Also included, but not reviewed here, are positive results from living fetal and adult human and animal brain tissue, living human and animal isolated brain cells, and postmortem human and animal brains. These experiments all provide evidence supporting the conclusion “that brain structure and even many brain functions are likely to be reasonably well preserved by freezing in the presence of cryoprotective agents,” and ushering in a new era of research in cryopreservation of the brain.
MOVING BEYOND BASIC CRYOPROTECTIVE PROTOCOLS
In the earliest days of cryobiology (the 1950s), cells and very small tissue samples were cryoprotected by diffusion—literally, soaking the sample in glycerol or DMSO. But larger systems, such as organs and whole organisms, are difficult to cryoprotect in this way and cells are damaged by rapid exposure to high concentrations of cryoprotectant. It was quickly recognized that the first obstacle can be overcome by utilizing the circulatory system of an organ to more rapidly introduce cryoprotectants to cells (i.e., perfusion). Osmotic shock can be overcome to some degree by introducing the cryoprotectant solution in a controlled fashion, starting with a low concentration and gradually moving to higher concentrations, and toxicity can be reduced by doing these procedures at low temperatures.
There were many reasons to believe that results from tissue and organ experiments could be applied to whole organisms, so Alcor began employing such a “cryoprotectant ramp” protocol in cryonics cases in the 1980s. Prior to that glycerol was introduced rapidly, resulting in a shorter perfusion and ending in a lower terminal concentration of cryoprotectant. Upon recommendation of a respected cryobiologist, Alcor also decided to increase the concentration of glycerol to 7.5M, which constitutes a practical limit in terms of viscosity and perfusion times. In 1993, Darwin et al. performed a series of experiments to validate the new protocol’s effectiveness in improving cryoprotection (and minimizing freezing damage) of patients.
In these experiments, the researchers carried out simulated cryonics cases on dogs, followed by cooling and storage at -90°C. A cryoprotectant ramp was used in one group of dogs, while another group was perfused according to an older protocol. After 18 months, the dogs were rewarmed and brain samples were examined using light and electron microscopy.
In general, a higher degree of ultrastructural preservation was observed in the brains of dogs that underwent a longer, gentler perfusion of cryoprotectant. Though there was still evidence of damage in these animals, it was considerably less than that observed in those treated with the simpler protocol. The results were published in the article “Effect of Human Cryopreservation Protocol on the Ultrastructure of the Canine Brain” (Darwin et al., 1995), in which very detailed descriptions and photographs are provided. This article may be found on the Alcor website.
IMPROVED TECHNOLOGIES AND CRYOPROTECTANTS
In the meantime, progress towards icefree cryopreservation of cells, tissues, and organs (including the brain) was made during the 1980s and 1990s. Most notable was Dr. Gregory Fahy’s development of vitrification as an approach to cryopreservation. Vitrification, which means “turning into a glass,” occurs when water is cooled too fast to form ice crystals. Fahy proposed that vitrification can also occur when a tissue is loaded with so much cryoprotectant that the entire volume of the tissue becomes a glassy solid during cooling, without any freezing at all. The advent of vitrification was a major leap forward in cryopreservation technology, and Alcor eventually implemented this approach in cryonics cases.
Though it eliminates mechanical freezing damage caused by ice crystals, the very high concentrations of cryoprotectant necessary for vitrification are toxic to cells. Over time, the composition of cryoprotectant solutions has also changed considerably from monoagents like glycerol to solutions consisting of multiple cryoprotectants, polymers, and synthetic “ice blockers.” Much of this work was done with the goal of reducing cryoprotectant toxicity and relaxing the cooling rates necessary to vitrify and rewarm complex organs.
Vitrification as an approach to cryopreservation of various cells and tissues has now been validated in numerous experiments and peer reviewed papers. Of greatest interest to cryonics is a study published by Pichugin, Fahy et al., in 2006. In their paper, “Cryopreservation of rat hippocampal slices by vitrification,” the researchers vitrified thin slices of rat brain using an advanced vitrification solution, VM3, containing penetrating cryoprotectants, non-penetrating polymers and “ice blockers” in a carrier solution designed to maintain viability and mitigate chilling injury. Slices treated with VM3 showed excellent ultrastructural and histological preservation after vitrification as compared to frozen-thawed slices. But, more importantly, the previously VM-3 vitrified tissue also exhibited a K+/Na+ ratio in the same range as control (untreated) slices, providing evidence of retained cellular viability. The vitrification solutions that have been used by Alcor to date (B1C, B2C, and M22) reflect the discoveries that have been identified in this and other 21st Century Medicine, Inc., papers.
This progress in cryopreservation of brain slices goes a long way toward establishing the credibility of cryonics as a legitimate scientific and medical endeavor, but an even more convincing statement could be made if we are able to provide evidence of functional recovery of previously vitrified brain tissue. Such evidence would include recovery of spontaneous and/or organized neural activity, or maintenance of a previously trained neural response, such as long-term potentiation (LTP). Starting at the first Suspended Animation conference in May 2007 researchers at 21st Century Medicine, indeed, began disseminating preliminary results showing that organized neural activity has been recovered in previously vitrified brain slices.
But even successful observation of LTP after cryopreservation provides only indirect evidence for memory maintenance. Alternatively, postburst after hyperpolarization (AHP) of hippocampal CA1 neurons may be characterized after cryopreservation of animals that have successfully acquired a hippocampal-dependent (memory) task. The demonstration of reduced AHP and accommodation in hippocampal neurons after acquisition of such a task and subsequent cryopreservation of the brain would be a huge step in the direction of proving that memories can be cryopreserved.
Beyond slices, recovery of whole brain electrical activity (EEG) after vitrification and storage at cryogenic temperatures would further provide strong empirical evidence that cryopreservation is a means of saving human lives. In 2012 my own company, Advanced Neural Biosciences (ANB), developed, to our knowledge, the first small animal EEG model for cryobiology research and we have been successful in recovering EEG activity after cooling and rewarming from 0°C. Our next, and more difficult, challenge will be to extend these results to high-subzero temperatures, and ultimately, to cryogenic temperatures.
CHALLENGES IN BRAIN CRYOPRESERVATION
While there have been significant advances in cryonics over the last 40 years, there also remain significant barriers to meeting the goal of reversible brain cryopreservation. Among the most important to address include cryoprotectant toxicity, brain dehydration, fracturing, and ischemia.
When a cryoprotectant solution is introduced to tissues, more than 50% of the water inside of cells is replaced by cryoprotectant molecules (which cannot freeze). Such a high concentration of cryoprotectant, while preventing freezing, comes with its down-sides. Cryoprotectant toxicity, discussed briefly earlier, is the tradeoff for eliminating the mechanical damage caused by freezing via the use of high concentrations of cryoprotectants. Add low temperatures to the equation and many of the things we know about the toxicity of such agents at ambient temperatures donâ€™t apply anymore, making the situation that much more complicated.
What we now know is that higher lipophylicity correlates with higher toxicity, as does strong hydrogen bonding (probably by disrupting the hydration shell around macromolecules). Other important discoveries include the reduction of toxicity when two highly toxic cryoprotectants are combined, such as DMSO and formamide. In general, however, our understanding of the mechanisms of cryoprotectant toxicity remains incomplete.
Brain dehydration following cryoprotection is another vexing issue. Dehydration of the brain is observed in cryonics in cases when cryoprotective perfusion is started and the blood-brainbarrier (BBB) is still intact. Because it is only seen when circulatory access is uncompromised, brain dehydration is often thought of as an indicator of a “good perfusion.”
The BBB, a network of endothelial cells forming tight junctions around capillaries, functions to separate the circulating blood from the brain extracellular fluid and thereby prevent unwanted and potentially dangerous bacteria and molecules from entering the brain. Unfortunately, that includes some components of cryoprotectant solutions, and the osmotic imbalance this causes during perfusion can result in dehydration. In good cryonics cases, dehydration of up to 50% total brain volume has been observed.
It is currently not known what degree of dehydration still permits recovery of function in the brain, but research by Yuri Pichugin at the Cryonics Institute suggests that opening the blood brain barrier may permit higher viability of brain slices after cryoprotective perfusion of the whole brain. Intuitively, however, severe dehydration of the brain would be preferable to avoid if possible, especially since we don’t know how much dehydration is compatible with reversal.
Another well-known issue in the cryopreservation of mammalian organs and human cryopreservation is fracturing, or “cracking,” caused by thermal stress during cooling. Thermal stress occurs because different parts of the tissue cool at different rates, resulting in different rates of thermal contraction. In vitrified samples, fracturing occurs mostly between the glass transition temperature (the temperature at which the cryoprotectant solution and tissues vitrify, around -120°C) and liquid nitrogen temperature (-196°C).
Alcor has observed fractures in the bodies and brains of patients removed from cryopreservation either by court order or for transfer and conversion to neuropreservation. Measurement of fracturing events using an acoustical monitoring device has enabled Alcor to plot events during the cooling process. In “Systems for Intermediate Temperature Storage for Fracture Reduction and Avoidance,” Brian Wowk reports that “acoustic events consistent with fracturing were found to be universal during cooling through the cryogenic temperature range. They occurred whether patients were frozen or vitrified. If cryoprotection is good, they typically begin below the glass transition temperature ( 123°C for M22 vitrification solution). If cryoprotective perfusion does not go well, then fracturing events begin at temperatures as warm as 90°C.” Preliminary inspection of fracturing events at Alcor suggests the newer generation of vitrification agents produce fracturing at lower temperatures than the older (glycerol) protocols, with the lowest first fracturing temperature being recorded at -133°C.
One approach to limit or eliminate fracturing is Intermediate Temperature Storage (ITS) in which the patient is stored at a temperature (slightly) below the glass transition temperature of the vitrification solution but above liquid nitrogen temperature.
Finally, ischemia remains a major obstacle to successful cryopreservation. Ischemia, or a lack of blood flow to tissues, is experienced globally (throughout the whole body) when the heart stops, as is legally required for cryonics procedures to begin. This lack of blood flow kicks off a biochemical chain reaction known as the “ischemic cascade” ultimately leading to cell death and tissue deterioration. And while some terminal patients may have a cryonics standby team at the bedside to minimize ischemic damage, many others are not so lucky. Some cryonics members die alone and may experience several hours, or even days, of ischemia at ambient temperatures. Even in cases with rapid stabilization there can be substantial periods of cold ischemia prior to the patient arriving at Alcor.
In a series of experiments in the early 1990s, Darwin et al. examined histological, ultrastructural, and gross structural preservation of the brains of both non-ischemic cats and cats that underwent 24 hours of warm and cold ischemia (30 min normothermic / 24 hours water ice temperature) prior to cryopreservation with glycerol. Compared to non-ischemic animals, cats perfused following ischemia exhibited significant perfusion impairment (areas of the body and brain that were not perfused at all) and more fracturing.
Experiments at Advanced Neural Biosciences certainly corroborate the above findings, and add to them. Over the past several years, we have extensively investigated the effects of both warm and cold ischemia on cryoprotection and cryopreservation of rat brains. In general, we have found that ischemia is positively correlated with perfusion impairment and ice formation (mostly in non-perfused areas). Cold ischemia, we have found, is much more amenable to intervention than warm ischemia, especially if the patient’s blood is replaced with an organ preservation solution such as Alcor’s MHP-2 — our best performing solution to date. We also found some intriguing preliminary evidence that perfusion with a high viscosity cryoprotectant was successful in (partially) overcoming ischemia-induced perfusion impairment. Having said all this, extended periods of warm and cold ischemia are not compatible with maintaining viability of the brain, no matter how good the cryoprotectant is. One potential solution is to introduce “field vitrification” in which the vitrification solution is introduced at a remote location and the patient is shipped on dry ice to the cryonics facility for further cool-down.
Another challenge in the practice of cryonics has been the lack of feedback concerning the effectiveness of cryoprotection protocols in human cases. Alcor has recently begun to take advantage of imaging technologies in order to assess cases. The Alcor CT Scan Project kicked off in December 2011 with the scans of two neuropatients (one cryoprotected and one “straight frozen”). Such scanning can provide valuable feedback regarding the quality of perfusion and cryoprotection of the brain and enables the direct comparison of brains cryopreserved after various patient scenarios such as immediate stabilization vs. long periods of warm or cold ischemia.
Similarly, Alcor would like to obtain very small brain biopsy tissue samples from patients prior to cryogenic cool-down and long-term storage. One pending question is whether CT scans and brain biopsies should be done routinely for all patients, and, if so, whether this should be on an opt-in or optout basis.
INTO THE FUTURE
Brain and whole body cryopreservation research is ongoing both at 21st Century Medicine (21CM) and Advanced Neural Biosciences (ANB). At 21CM, an ambitious research program aimed at understanding and improving brain preservation is currently in progress. Among the issues that 21CM are studying are the ideal composition of cryoprotectants and carrier solutions for the brain, the relationship between perfusion protocols and ultrastructure, and the effects of various methods to open the blood brain barrier. While 21CM has made a number of important discoveries in brain preservation, the publication of a new peer reviewed paper on this topic is pending the resolution of proprietary issues.
At ANB, we are currently utilizing electroencephalography (EEG) in rats to assess the potential for functional recovery of the whole brain after cryopreservation. As discussed, we are taking an incremental approach, first cooling and resuscitating rats from temperatures just below those causing cardiac arrest (around 25°C), then from 0°C, and later from increasingly lower subzero temperatures requiring introduction and removal of cryoprotectant.
Our whole brain cryopreservation research uses two distinct models: (1) whole body resuscitation and (2) isolated brain perfusion. We aim to push the whole body model down to the lowest temperatures possible but will switch to the isolated brain (or head) model when we reach the (shortterm) limits of that model. The advantage of the whole body resuscitation model is that we not only recover whole brain electrical activity but heart and respiratory function, too. Like 21CM we are also seeking a better understanding of the effects of brain dehydration on viability because we expect that severe dehydration will become a limiting factor in recovering brain function after cooling to cryogenic temperatures, and perhaps even at high subzero temperatures.
Although both Alcor and the Cryonics Institute introduced their most recent vitrification solutions in 2005 and the last peer reviewed journal article about brain cryopreservation was published in 2006, research in this area continues and progress is undeniably being made. Building upon observations of good structural preservation of brain cells and tissues in the 70s and 80s, the goals for such research programs are well-defined and involve obtaining ever-better structural preservation and the recovery of viability and, ultimately, recovery of function after cryopreservation of whole brains.
The accomplishment of each of these goals could provide more evidence to uphold the argument that cryonics patients are not dead by contemporary medical criteria and helps to validate cryonics as an evidencebased procedure for preserving terminally ill patients in a state of suspended animation until they can be successfully treated. More than any other advances in cryobiology to date, the functional recovery of a whole brain, either isolated or within an organism, will allow cryonics organizations to stand on solid scientific ground and to focus more energy on improving preservation methods for at-risk patients and less energy on arguing whether cryonics is a viable technology.
A timeline is provided below to identify research and developments that have contributed to progress in brain cryopreservation.
CHRONOLOGY OF PAPERS AND DEVELOPMENTS
RELEVANT TO BRAIN CRYOPRESERVATION
Suda I, Kito K, Adachi C. “Viability of long term frozen cat brain in vitro,” Nature, 212, 268-270 (1966).
Suda I, Kito K, Adachi C. “Bioelectric discharges of isolated cat brain after revival from years of frozen storage,” Brain Research, 70, 527-531 (1974).
Fahy GM, Takahashi T, Crane AM. “Histological cryoprotection of rat and rabbit brains,” Cryo-Letters, 5, 33-46 (1984).
Hixon H. “Getting to 8M glycerol and other problems,” Cryonics, 14(11), 21 (1993). (Transition of Alcor to high molar glycerol solutions.)
Darwin M., Russell S., Wakfer P., Wood L., Wood C. “Effect of Human Cryopreservation Protocol on the Ultrastucture of the Canine Brain.” Originally published by BioPreservation, Inc., as BPI Tech Brief 16 on CryoNet and sci.cryonics, May 31, 1995.
Chamberlain F. “Vitrification arrives: New technology preserves patients without ice damage,” Cryonics, 21(4), 4-9 (2000). (Introduction of vitrification solution for neuropatients at Alcor.)
Fahy GM, Wowk B, Wu J, Paynter S. “Improved vitrification solutions based on the predictability of vitrification solution toxicity,” Cryobiology, 48(1):22-35 (2004).
Lemler J, Harris SB, Platt C, Huffman T.“The Arrest of Biological Time as a Bridge to Engineered Negligible Senescence,” Annals of the New York Academy of Sciences, 1019, 559-563 (2004).
“New Cryopreservation Technology,” Alcor website (2005). (Introduction of M22 for both neuropatients and whole body patients at Alcor.)
Best B. “The Cryonics Institute’s 69th Patient,” Cryonics Institute website (2005). (Introduction of VM-1 at the Cryonics Institute)
Pichugin Y, Fahy GM, Morin R,“Cryopreservation of rat hippocampal slices by vitrification,” Cryobiology, 52, 228-240 (2006).
Announcement of recovery of organized electrical activity after vitrification of brain slices by 21st Century Medicine, Suspended Animation Conference, Fort Lauderdale, Florida.
Advanced Neural Biosciences establishes small animal EEG model for cryobiology research.
Fahy GM, Guan N, de Graaf IA, Tan Y, Griffin L, Groothius GM. “Cryopreservation of precision-cut tissue slices,” Xenobiotica, 43(1):113-32 (2013). Abstract. (Examination of precision-cut tissue slices of vitrified rabbits, including the brain.)