Science | Institute for Evidence Based Cryonics

Resuscitation and Reintegration of Cryonics Patients Symposium

29. April 2013 · Comments Off · Categories: Cryonics, Science, Society

On Sunday May 12, 2013, the Institute for Evidence Based Cryonics will organize a symposium about the resuscitation and reintegration of cryonics patients in Portland, Oregon. To our knowledge, this is the first public meeting exclusively concerned with the repair, resuscitation, and reintegration of cryonics patients.

The symposium is being held at The Cleaners at Ace Hotel (The Cleaners at Ace Hotel 403 SW 10TH AVE, 97205) in downtown Portland, Oregon from 10:00 am to 07:00 pm.

Admission is free. Registration for the event is possible at the event Facebook page.

On Saturday evening, the day prior to the symposium, Aubrey de Grey and Max More will be speaking about rejuvenation biotechnologies and cryonics at the Paragon Restaurant & Bar in Portland, Oregon.

Admission for this event is free and registration for this event is possible on the event Facebook page, too.

The current line-up of speakers is as follows (the exact schedule will be announced soon):
BEN BEST – EFFECTS OF TEMPERATURE ON PRESERVATION AND RESTORATION OF CRYONICS PATIENTS

Macromolecular temperature is a quantification of atomic-level molecular motion. The ability to maintain and reconstruct cryonics patients could be critically dependent on low temperature atomic/molecular motion and on the ability to operate nanomachines at cryogenic temperatures. Possible problems and solutions will be discussed.

Bio: Ben Best was President of the Cryonics Society of Canada for about a decade, after which he was President of the Cryonics Institute for nearly a decade. He is currently Director of Research Oversight for the Life Extension Foundation. The cryonics section of his website is one of the best sources of information about the science behind cryonics available on the internet ( www.benbest.com/cryonics/cryonics.html )

CHANA DE WOLF – RECONSTRUCTIVE CONNECTOMICS

Complete preservation of the “connectome” should be sufficient for meaningful resuscitation attempts of cryonics patients but it may not be necessary. As long as the original connectome can be inferred from what is preserved, damage associated with cerebral ischemia or suboptimal cryonics technologies do not necessarily exclude future resuscitation. In this presentation I will present a general framework for reconstructive connectomics and explore theoretical and experimental research directions for reconstructing damaged and altered connectomes.

Bio: Chana de Wolf lives in Portland, Oregon, where she works as a business manager and biomedical researcher. She holds a B.S. in Experimental Psychology (2001), an M.S. in Cognition and Neuroscience (2003), and has extensive management and laboratory experience. She has several years of experience working as a research assistant in a variety of laboratory environments, and has taught college-level courses in neuroscience lab methods and biology. She is a Director and researcher for Advanced Neural Biosciences. Chana joined as a member of the Alcor Life Extension Foundation in 2007 where she also worked as a Research Associate at Alcor to help build a sustainable, multi-faceted cryonics research program

RANDAL KOENE – BRAIN EMULATION AND NEUROPROSTHETICS: A SYSTEM OF FUNCTIONS TO BE SUSTAINED

Being, now or following revival from cryopreservation, ultimately depends on one’s ability to experience and to do so in the manner that is characteristic of one’s individual mind. Recently, it has become possible to address this problem in a concrete and systematic manner, largely due to rapid advances in computational neuroscience and data acquisition, both structurally (the popular field of “connectomics”) and functionally (brain activity mapping). The process of personal experience – like any process – involves some mechanisms operating at a given time under the influence of an environment state, a state that can include sensory input and functional “memory” established as a result of prior conditions. An emulation or prosthesis is then the attempt to replace a system of processing with an equivalent set of mechanisms that carry out the same processing within established success criteria. The engineering approach to understanding a system sufficiently that it can be emulated or replaced by prostheses is known as system identification. I will describe how system identification may be feasibly carried out for an individual human brain, and how constraints and requirements can be learned through projects with iterative improvements. I will present the projects that are underway to develop neuroscience tools with which successful system identification may be accomplished.

Bio: Dr. Randal A. Koene is CEO and Founder of the not-for-profit science foundation Carboncopies.org as well as the neural interfaces company NeuraLink Co. Dr. Koene is Science Director of the 2045 Initiative and a scientific board member in several neurotechnology companies and organizations.

MAX MORE – MAXIMIZING REVIVAL PROBABILITY: PRESERVATION, RECORDING, INTERPOLATION, AND RECONSTRUCTION

The proper ultimate goal of cryonics is reversible suspended animation. While we should continually strive for that goal, we do not know if or when it will be fully achieved. Until then, we must grapple with the probability that cryopreservation will in itself not fully preserve personal identity critical information. A revived individual may be missing pieces of his or her life, or some of the existing pieces may be fuzzier than they were before clinical death. It may be feasible to fill in the gaps and to sharpen the focus by feeding into the repair and revival process biographical information with a high degree of resolution. That information may also serve to validate the accuracy of a reconstructed connectome. Up to the present, cryonics organizations have offered minimal storage of personal-identity relevant information. In this talk, I will consider ways in which members of cryonics organizations could use the emerging tools and technologies associated with the “Quantified Self” concept to capture and record detailed biographical information, and how cryonics organizations could assist with this and convey the resulting data to a future capable of repairing and resuscitating cryonics patients.

Bio: Max More is the President & Chief Executive Officer of the Alcor Life Extension Foundation. More has a degree in Philosophy, Politics, and Economics from St. Anne’s College, Oxford University (1984-87). He was awarded a Dean’s Fellowship in Philosophy in 1987 by the University of Southern California. He studied and taught philosophy at USC with an emphasis on philosophy of mind, ethics, and personal identity, completing his Ph.D. in 1995, with a dissertation that examined issues including the nature of death, and what it is about each individual that continues despite great change over time.

KEEGAN MACINTOSH – REINTEGRATION OF CRYONICS PATIENTS: LEGAL AND LOGISTICAL CONSIDERATIONS

Given the host of complicated problems to be solved before resuscitation of cryonics patients is possible, it is easy to leave planning for their reintegration for another day. However, this assumes that there is nothing particularly important that can be done about reintegration prior to patient cryopreservation, which might be impossible, or at least far more difficult afterward. It also underestimates the impact that fear of dis-integration has on individuals’ decisions on whether to sign up for cryonics, which might be alleviated if we had more concrete plans for reintegration, with presently actionable components. In this talk, Keegan Macintosh will survey several aspects of cryonics patient reintegration, both legal and logistical, that can be tangibly worked on today.

Bio: Keegan Macintosh received his J.D. from the University of British Columbia in 2012, and is Executive Director of the Lifespan Society of British Columbia, a non-profit organization established to educate the public on life extension strategies and protect access to potentially life-saving technologies. Keegan is a board member of the Institute for Evidence Based Cryonics, as well as the Cryonics Society of Canada.

ASCHWIN DE WOLF – CRYONICS WITHOUT REPAIR

Cryonics aims to stabilize critically ill patients at low temperatures in anticipation of future medical treatment. While the concept of cell repair is often associated with the practice of cryonics, it is not an intrinsic element of the procedure. Advanced cryonics technologies will permit reversible cryopreservation of the patient. If human suspended animation would be achieved cryonics would solely involve future treatment of the patient’s disease and its underlying pathologies. In this talk I will discuss why reversible cryopreservation is important and which technical obstacles need to be overcome to make it a reality.

Bio: Aschwin is a Director and researcher for Advanced Neural Biosciences, the editor of Cryonics magazine, serves as a consultant for a number of cryonics organizations, and has published technical articles on various cryonics topics.

In praise of cold

14. January 2013 · Comments Off · Categories: Cryonics, Neuroscience, Science

Some observers believe that cryonics advocates are reluctant to subject their theories to experimental scrutiny because this could damage their (uncritical) belief in future resuscitation. Similarly, one might think that cryonicists would react with a mix of hostility and dismissal to alternative strategies for personal survival. Nothing could be further from the truth. In fact, it is exactly because our personal survival is at stake that forces us to be wary of dogmatism.

For this reason, I have always been interested in chemical fixation as a (low cost) alternative for cryonics. In fact, years before all the talk about the “connectome” and “plastination” I spent considerable time exchanging messages with Michael Perry at Alcor about the technical and practical feasibility of chemical brain preservation. But no matter how open minded I tried to be about this approach, I kept running into the same challenges over and over again.

The challenge that has concerned me the most is whether a delayed start of chemical brain fixation will produce incomplete distribution of the chemical fixative in the brain because of ischemia-induced perfusion impairment. Thinking about the technical problem of “no-reflow” is not the first thing on the mind of someone who first hears about the idea of using chemical fixatives to preserve the brain. In my case, this concern was not just “theoretical.” In my lab I have spent many years looking at the effects of cerebral ischemia on cryopreservation and chemical fixation. Last year we decided to broaden our investigations to delayed chemical fixation and we have not been pleased at what we have observed so far. After 1.5 years of room temperature storage the delayed aldehyde fixed brains are falling apart and continue to decompose. In small animals one might imagine that such perfusion impairment could be overcome by immersing the brains in the fixative instead but human brains are simply too large. By the time that the fixative would have reached the core of the brain, extensive autolysis will have occurred.

Another complex problem is to identify a fixation and polymerization protocol that fixes all identity-critical parts of the brain. If aldehydes do not completely fix the lipids in the brain, should we add strong oxidizing heavy metals to stabilize lipids? This is possible in theory but, as a general rule, these chemicals are either very expensive or dangerous to use (or both). Even if we are able to identify a chemical fixation protocol for the brain that can do the job, how can we know that such brains are stable for very long periods of time? Should we follow fixation by embedding with a polymer to inhibit residual biochemical activity? To my knowledge, there is no known embedding protocol that is scalable to human brains due to the extreme viscosity of these plastics.

Recently these issues took a more personal nature for me when I had to think really hard about a reasonable but affordable longterm preservation protocol for a companion animal. I spent many days reading the electron microscopy and fixation literature to come up with a protocol that was better than aldehyde fixation and low temperature storage. Adding calcium to the fixative? What about phenol? Post-fixation perfusion of a viscous cryoprotectant to allow storage at subzero temperatures? That is when I really started appreciating the “magic” of cold temperatures.

Absent a vitrification agent, cryogenic temperatures can cause extensive damage to cells. But one thing we know: whatever the nature of this damage, as soon the brain is below the glass transition temperature of -130°C, all water is either frozen or a vitrified rigid solid. We do not have to worry about any damage getting worse over time, or whether some biomolecules have not been fixed. Cold may be “crude” in its effects but it is exactly because no biochemical process can escape inhibition at very low temperatures that makes it such a powerful personal survival technology.

Originally published as a column (Quod incepimus conficiemus) in Cryonics magazine 2013-1

Preserving and inferring

17. October 2012 · Comments Off · Categories: Cryonics, Science

A common complaint against cryonics is that existing cryopreservation technologies may not be good enough to preserve the ultrastructure of the human brain. Advocates of cryonics often object that such views do not reflect actual inspection of the evidence of cryopreserved brains but instead reflect misconceptions about “freezing” and ice formation rupturing cells. But the more fundamental misconception rests on the view that for cryonics to work flawless preservation of the brain is absolutely essential.

This view is not only mistaken but holds cryonics to higher standards than those applied in conventional medicine. In medicine it is routine for patients to present themselves with conditions in which an organ or tissue has been changed from its normal condition (or appearance) as a result of disease or trauma. Restoring normal structure or function is the essence of most medical treatments.

One might object that in the case of cryonics we are concerned with the brain, which distinguishes itself from other organs that encodes highly individual information. If a portion of the brain is erased we cannot consult another brain or medical textbook to infer its original state. We can admit that this is a valid observation but it is not necessarily a fatal argument against cryonics, provided the damage has not reached the point of complete destruction or indecipherability.

There is a difference between damage and obliteration. If we look at electron micrographs of brain tissue produced at various points in time after circulatory arrest (“death”) we will observe progressive alterations of synapses, cell membranes, organelles etc. We describe such changes with a mental (or actual) map of how they normally look like in mind. At this level the fact that the brain is a highly individual organ is no longer relevant because we know the universal biochemical language in which this identity is written. At this point the real question becomes at which point is it not even possible to infer the original condition of the brain. As far as we understand this today, this may be a question of many hours, if not days.

This robustness of identity-critical information in the brain may seem to contradict the routine observation in emergency medicine that there is a much narrower time limit for successful resuscitation from cardiac arrest. The crucial difference here is that we are no longer talking about the ability to infer identity-critical information but restore physiological function. But function is a lot more vulnerable to metabolic and biochemical changes than the wiring of the brain. In fact, if function were a necessary requirement to infer information a lot of existing forensic and archeological science would be impossible.

In approaching cryonics it is important to recognize the distinction between preserving and inferring. In this way we can better assess the prospects for resuscitating patients who were cryopreserved under nonideal conditions and/or with older technologies.

Originally published as a column (Quod incepimus conficiemus) in Cryonics magazine 2012-5.

ApoE4, diet, and gender

25. July 2012 · Comments Off · Categories: Health, Science

As we learn more about the human genome, there will be an increasing recognition that general diet recommendations are going to give way to diet recommendations that more closely track the genotype of individuals. For those interested in healthy life extension an important question concerns the relationship between ApoE status and diet. In Why We Age : What Science Is Discovering About the Body’s Journey Through Life (1997) Steven N. Austad writes:

.. piles of evidence suggest that certain genes have a major impact on the development of atherosclerosis, probably the major disease of aging in the Western world. One of those genes is the Apolipoprotein E, usually abbreviated ApoE, which is involved in processing dietary fat. People with one form of the gene, called e4, have higher blood cholesterol (as well as higher LDL, or ”bad” cholesterol) levels than people with other forms of the gene. Finns have the highest rate of atherosclerosis in the world and also have one of the world’s highest frequencies of e4. The Japanese have the world’s lowest national rate of atherosclerosis and also among the world’s lowest frequency of e4. So you could call e4 an atherosclerosis gene. But this would be misleading, because the world’s highest frequency of e4 is found in a country, Papua New Guinea, where until recently atherosclerosis was virtually unknown.

People living in the bush in Papua New Guinea eat a low-fat diet (less than 5 percent fat, compared with 30 to 40 percent fat in an American diet) from necessity rather than choice. Their daily life also involves exercise at levels that would cripple or kill most Americans, even the athletically inclined….So genes operate not in a vacuum but in a specific environment. This is something to bear in mind when reading of the discovery of new “longevity” genes. For instance, there is another form of the ApoE gene, e2, which appears to lower blood cholesterol and therefore probably protects against developing atherosclerosis. Is this a longevity gene? It depends on the environment. Where people eat a lot of fat and don’t exercise, it may well be a longevity gene. In fact, French centenarians are about twice as likely to have this gene as the French population as a whole. But in other environments, the gene may well have little or no effect.

What these examples suggest, besides the difficulty in defining genes with respect to longevity, is that unless we understand how a particular gene is influenced by a particular environment, it will be difficult to translate the effects of genes from animals to humans. This is why most gerontologists are hesitant to claim too much about the relevance to humans of genes now being found with increasing frequency in simple organisms such as fungi and worms that seem to slow aging dramatically. It is difficult to draw parallels between human and worm and fungal environments. (p-41-43)

It is important to keep this point in mind when one considers the pro- and cons of a popular diet. For example, the Paleo Diet has become increasingly popular in the life extension & transhumanist communities. But if the observations of Austad are correct, a diet high in (saturated) fat could have adverse consequences for carriers of one or two copies of the ApoE4 gene. In fact, in her book The Perfect Gene Diet Pamela McDonald steers ApoE4 carriers in the direction of a vegetarian / vegan diet. As we learn more about the ideal diet for carriers of the ApoE4 gene, further refinements may be expected.

Another interesting emerging finding about ApoE4 is that its effect on having a higher probability of developing late-onset Alzheimer’s disease may be dependent on gender. A number of preliminary studies have found that the risk for developing Alzheimer’s disease for males with just one copy of the ApoE4 gene may not be much different from that of individuals who carry the more common ApoE3 gene:

Together with the previous meta-analysis, the data support the idea that a man with one E4 allele may not have much more risk of AD than an E3 homozygote, Greicius said, but added, “If you have two copies of the E4 allele, whether you are a man or a woman, there is no question that your risk leaps tremendously.” He is analyzing older datasets to see if the interaction between gender and ApoE genotype holds, and is also looking for genes that act synergistically with ApoE in women but not men.

If there is anything that is becoming clear from such studies it is that it will be increasingly inadequate to make sweeping statements about lifestyle, diet, and longevity without taking into account ethnicity, gender, age, genotype, and environment of a person. This does not mean that all general recommendations should be discarded. For example, there could be good reasons to believe that a low calorie diet and (moderate) exercise benefit most people. But when it is comes to the nitty gritty of what to eat and how to exercise a more personalized approach is warranted.

The 2012 Society for Cryobiology Meeting

21. June 2012 · Comments Off · Categories: Cryonics, Science

From June 3 to 6, 2012 I attended the annual Society for Cryobiology meeting, which in 2012 was held in Rosario,Argentina.

Attending with Argentine biogerontologist and Cryonics Institute Member Rudy Goya (who was profiled on page 6 of the Nov/Dec 2011 issue of LONG LIFE magazine) may have reduced the interaction I had with the cryobiologists. There were fewer sessions than usual, and thus more free time. The welcome reception was not held until the evening of the first day of the sessions.

The first session dealt with an aspect of Argentine cultural heritage, the Llullaillaco children — three Inca children who had been mummified by dehydration high on a volcano and preserved for over 500 years. Two of children were selected by the Incas because they were “perfect” (beautiful and pure) at 6 or 7 years of age. It was believe to be an honor to go directly to heaven, not really death or sacrifice. The children were given an intoxicant and buried alive atop the Llullaillaco volcano. Much of the session focused on the conditions that caused the children to be so well-preserved, and the conditions the curators should use to preserve the children for the future — involving careful regulation of temperature, atmosphere, humidity, and an environment inhospitable to most microbes.

If reanimated cryonicists receive anything like the care these children are receiving, there should be no concerns about being welcome in the future. In a sense, the Incas had it right when thinking they were sending the children to heaven. Of course the Inca children were deprived of life and are unable to experience or enjoy their treatment by modern curators — and cryonicists should not encourage hastening death based on reliance on unproven future technologies.

At this conference there were special “How to do it?” sessions overlapping part of the lunch hour that focused on practical techniques unrelated to the experimental results and theoretical considerations covered in the regular sessions. Sunday’s topic was proteomic analysis, which covered removal, isolation, and identification of proteins from cells. The presenter (from the Institute of Molecular Cell Biology in Rosario) claimed that instrumentation allowing high throughput and resolution had given proteomics a maturity comparable to genomics.

The afternoon sessions were concerned with cell and tissue preservation. Elza Cabrita reported on improved cryopreservation of fish sperm through a combination of cryoprotectants and antioxidants. Locksley McGann reported on experiments sequentially exposing human articular cartilage to four CPAs (DMSO, glycerol, propylene glycol, and ethylene glycol) at lowering temperature (0ºC, −10ºC, −15ºC). Vitrified samples were cooled to liquid nitrogen temperature, and demonstrated 75% cell recovery when rewarmed.

Adam Higgens reported on an improved procedure for washing glycerol from red blood cells. Currently about 99% of banked blood is stored at refrigerator temperature (2-4ºC), with a shelf life of 42 days. Only 1% of blood (mostly rare blood types) is cryopreserved with glycerol and stored at −80ºC, with a shelf life of ten years. A major deterrent preventing more blood from being banked at −80ºC is the 30-60 minute glycerol washout procedure. Adam’s group developed a procedure that can wash the glycerol out in 30 seconds, but 5 seconds longer or shorter results in too much hemolysis. A three minute washout procedure is less time sensitive (one minute longer or shorter is tolerable), but the method needs to be scaled-up from the 0.5 milliliter test volumes being used.

On Monday, Peter Mazur reported that in vitrifying mouse oocytes, it is the warming rate and not the cooling rate that is most critical for success. He spoke of microwave warming and the problem of thermal runaway (uneven warming). Ice blockers would not cross cell membranes, and thus would not be of use against intracellular ice formation. Pier Morin reported on miRNA microarray assessment of miRNA expression of the freeze-tolerant insect goldenrod gall fly at control (+5ºC) and freezing (−15ºC) temperatures. mIR-210 was down-regulated and mIR-1 was up-regulated at freezing temperature (the latter is involved in cell cycle regulation).

Ali Eroglu reported on epigenetic perturbation resulting from human oocyte cryopreservation techniques. Both the slow freezing and vitrification methods he used resulted in down-regulated expression of H19 and Ube3a genes. Igf2r was down-regulated by vitrification, but not by slow freezing.

Monday’s “How to do it?” session described a combination of nanotechnology and stem cells for tissue engineering. Specifically, electrospinning can be used to create a nanometer scale web of biodegradable fibers that can be populated with mesenchymal stem cells by electrospraying. The main challenge is vascularization of the tissue. Vascular Endothelial Growth Factor (VEGF) increases cell adhesion, but not necessarily vascularization.

Barry Fuller reported on successful hypothermic perfusion of liver. A kidney hypothermic perfusion machine has been in operation for ten years, but liver has been more challenging, because of its large size and the fact that two vessels supply the organ (hepatic artery and portal vein). The liver hypothermia perfusion machine uses two pumps.

PhD student Na Guan described her study of gene expression changes associated with chilling injury of rat liver slices. Cryoprotectant solutions supplied by 21CM (Greg Fahy) were used to ensure no ice formation interfered with the process. ATP levels indicated that the cryoprotectant solutions used were causing no damage, although the composition of those solutions was not disclosed. 1108 genes were observed, of which 251 were up-regulated and 77 were down-regulated by chilling at −15ºC. Focusing on the top ten up- and down-regulated genes: inflammatory and DNA repair genes were considerably up-regulated, and genes associated with biosynthesis of cholesterol and polyunsaturated fatty acids were down-regulated. The latter seems paradoxical in light of the up-regulation of cell surface-linked signaling pathways, which indicate cell membrane injury.

During the question period, both Andreas Sputtek and Arthur Rowe were sharply critical of the undisclosed composition of the 21CM cryoprotectant solutions being used. Sputtek said that because science is about disclosure of methods and materials, Guan’s work was not science. Guan said she had begged 21CM for disclosure, but said she was told that anyone wanting to replicate the experiments could buy the solutions from 21CM. Tiantian Zhang said that gene analysis only done 30 minutes after chilling injury does not give the whole picture. She said that in her own work doing gene analysis of fish oocytes or embryoes after chilling injury, gene expression changes dramatically with time — that it is a mistake to only analyze the expression 30 minutes after exposure as Guan had done. After the presentation, Arthur Rowe spoke with Guan telling her how much trouble he has had over the years with her collaborator (Dr. Fahy) in connection with the non-disclosure issue. I spoke with Guan myself after her presentation. She told me that the greatest chilling injury occurs at −90ºC. She also said that she would be getting her PhD in July and did not know who would be continuing her work. When I spoke to Dr. Fahy about the presentation, he told me that the composition of the vitrification solution had been disclosed and that Guan was mistaken in believing that she could not disclose the composition.

Tuesday morning had been scheduled to begin with a lecture by Ken Storey. Storey typically has no interest in what other cryobiologists have to say, is fairly ignorant of areas of cryobiology outside of hibernation and effects of low temperature on animals in nature, and only comes for his own presentation before leaving. His ignorance is on display when journalists get him to do cryonics-bashing, which he does with relish, but the general public only sees the comments of a respected cryobiologist, not the ignorant misunderstandings of cryobiology. I would not have expected Storey to come all the way to Rosario, Argentina only for his own presentation, but this is what he attempted to do — and he missed one of his flight connections. Ironically, this year Storey was honored by being made a Fellow in the Society for Cryobiology. Storey does, admittedly, have a fabulous knowledge of molecular biology, and is an outstanding scientist in connection with his own work.

To compensate for Storey’s absence the conference organizers arranged a makeshift follow-up session on the Llullaillaco children. This wasn’t entirely a waste, because many issues had not been addressed in the first round. I was going to question using a 2% oxygen and 98% nitrogen atmosphere for the children rather than pure nitrogen, but Barry Fuller raised this objection before I was called upon. I did, nonetheless, suggest that the goal should be to perfect the preservation environment rather than try to recreate the conditions of the mountain. Even this had not been done because the relative humidity had been raised to 70% on the bad advice of an expert rather than held to the 40% present on the volcano. The children were reportedly gaining 300 grams per year, probably from the humidity. There is a lower humidity limit below which no microorganisms can grow, but 0% relative humidity in the −20ºC preservation chambers would run the risk of freeze-drying.

For the second session on Tuesday, John Crowe had been scheduled to lead a symposium composed of 3 other speakers besides himself, but all of the other 3 speakers cancelled-out. John, nonetheless, did an excellent job of speaking for the whole session on the basis of his own work. John is an expert in dehydration and freeze-drying of organisms as well as on tardigrades and trehalose. Drying DNA with trehalose prevents fragmentation, and drying proteins with trehalose prevents denaturation. John discovered that drying liposomes with trehalose prevents membrane fusion — although he lost most of the patent rights on commercially valuable processes by publishing too soon. Dehydration of samples containing sucrose drives the glass transition temperature (Tg) from 20ºC to 60ºC, but dehydration of samples containing trehalose raises the Tg from 20ºC to 120ºC. More recently, however, it has been found that LEA proteins can be as protective as trehalose, but in a way that is distinctive and complementary to trehalose — stopping liposome fusion, preventing protein aggregation, and changing sample Tg. Yeast cells are protected against dehydration damage not only by trehalose, but by the trehalose transporter protein which exports the trehalose to the exterior membrane surface and imports the trehalose to the internal membranes of organelles such as mitochondria. But although the genome of tardigrades has been sequenced, the tardigrade trehalose transporter has not yet been identified.

Barbara Reed is probably the world’s foremost expert on plant cryopreservation, and she has spoken a lot about the benefits of antioxidants for cryoprotection. But the presentation Barbara gave on Wednesday gave me the strongest indication that oxidative stress could be a significant mechanism of cryoprotectant toxicity. Not only because a variety of cryopreserved patients show improved viability with Vitamin E, Vitamin C (if iron is removed), lipoic acid, glutathione, and melatonin — but because oxidative damage was shown to increase significantly associated with cryoprotectant loading.

Roland Fleck works with the UK Stem Cell Bank. The Bank conducted studies indicating that a 2-step freezing protocol results in better viability than vitrification. But examining the results of 8 technicians showed that in the hands of the most experienced technician vitrification was as effective as the 2-step freezing protocol. Protocols should not be so highly dependent upon technician expertise. After his presentation, Roland told me he was concerned that he was only able to assay viability by the use of trypan blue, which only indicates membrane integrity and does not provide a very fine measure of cell function. He said that the requirement to use the trypan blue viability assay was imposed by bureaucrats or scientists who do not have much knowledge of cryobiology.

Igor Katkov said that he believes any sperm cell can be vitrified simply by choosing the right cooling and warming rate. He said he was advised by his patient attorney to drop seven slides from his PowerPoint presentation.

At the business meeting the Society membership was reported to be down to 186. The journal CRYOBIOLOGY continues to be profitable. CRYOBIOLOGY has a 33% rejection rate, a 1.83 impact factor, and 33 Members on the Editorial Board. The Society has $300,000, which the IRS thinks is too much for a charitable organization, but the IRS is allowing the society to retain tax-exempt status. Increasing travel awards is the preferred use of money, but there is a problem that on the one hand travel awards are a taxable benefit, and on the other hand it is illegal to pay the taxes on travel awards. The 2013 conference (the 50th annual conference) is to be held in Washington, DC, where the first conferences were held. The 2014 conference might be Istanbul, Turkey and the 2015 conference could be in Isreal, but definite decisions have not been made.

Last year’s new Society for Cryobiology Fellows Barbara Reed and John Crowe each gave presentations reviewing their careers. Barbara Reed began as a plant biologist in 1985, but was brought into the field of cryobiology by a need to preserve germ tissue. John Crowe said that after the Sputnik shock of 1957 the US government sought to encourage more young people to go into science, including him. As a teenager, John was sent to a number of different science laboratories on his summer vacations. John considers himself more of a “dryobiologist” than a cryobiologist. He entertained us with photos taken in the many exotic countries he and and his wife have visited since his retirement.

The two new Fellows for 2013 are Ken Storey and Mehmet Toner.

This conference was attended by not more than about 80 people, at least half of whom were South America. There were maybe 30 or so hard-core Society for Cryobiology Members. This was my 9th annual meeting in a row, but for the most part I made little effort to relate to the cryobiologists, although one of my intentions in attending these meetings has been to soften the hostility of cryobiologists to cryonicists. I sat near the front of the meetings with Rudy who told me that he learned a great deal about the cryobiology behind cryonics practices by attending this conference. Very many of the cryobiologists were reporting on using vitrification at this conference, and including articular cartilage and plant tissue as well as single cells. I was fairly active in my questioning and comments — about which a few of the cryobiologists complimented me.

I lost my sense of urgency about talking to Peter Mazur. Peter recently told a journalist that although it is not possible to prove that the chance of cryonics patients being reanimated are zero, “you can, I think demonstrated that the probability of its being done is so extremely low that effectively it is zero” [CANADIAN MEDICAL ASSOCIATION JOURNAL; Monette, M; The Church of Cryopreservation; 184(7):749 (2012)] I am curious about the demonstration Peter has in mind, but I am also committed to learning from cryobiologists rather than arguing with them about cryonics. Peter walked away a few years ago when I asked him when solution effects rather than mechanical damage cause injury to cells due to slow cooling, so that may be a touchy subject with Peter as well.

I did, however, pepper John Crowe with questions — finding him to be friendly and informative. John confirmed what Peter Mazur had told me about cells being able to tolerate the loss of all osmotic water (freezable water, which constitutes at least 80% of cell water) without injury — a matter of great relevance in the vitrification of cryonics patients (assuming inter-cellular effects are not of great significance).

I sought-out Ali Eroglu, with whom I have had little interaction in the past, calling his attention to an article in the most recent issue of CRYOBIOLOGY about transfection of mammalian ovary cells with trehalose [CRYOBIOLOGY; Chakraborty,N; 64(2):91-96 (2012)]. Ali has microinjected oocytes with trehalose (along with low concentrations of DMSO to protect the mitochondria) [BIOLOGY OF REPRODUCTION; Eroglu,A; 80(1):70-78 (2009)]. Ali had not seen the CRYOBIOLOGY article, but he told me that ovarian tissue is easier to work with than oocytes.

At the final banquet I sat next to one of the conference organizers. He told me that John G Baust had been supposed to conduct a symposium, but had cancelled the whole thing a month before the conference without giving any explanation. He agreed with the comments I had made about the Llullaillaco children, and told me that a committee of cryobiologists was going to supplement the questionable advice that the Argentine government has been getting from a single advisor in New York. He told me that National Geographic had discovered the children and attempted to remove them from Argentina on a midnight flight, but the Argentine government got wind of the plan and intervened. Nonetheless, the children were simply kept in −20ºC freezers for several years while planning and building better preservation chambers.

The return bus trip to BA on Thursday took the entire afternoon — much longer than I would have expected. I sat next to Adam Higgens on the bus, and spoke with him much of the time, mostly about his life and work, as well as about our experiences in Argentina. Adam knew Spanish fairly well because he has spent four months of language immersion living in Equador (and visiting the Galapagos Islands). If he gets a patent for deglycerolizing blood, the University would get half the royalties and he would split his half with his collaborators. The advantages of his method would be the ten year rather than 42-day shelf life for banked blood, and the greatly reduced washout time. The latter is a significant savings in labor costs, but would have to be weighed against greater electrical costs for a −80ºC freezer as opposed to refrigeration. Even if he is successful in perfecting his methods, he thinks that the blood banking industry is too conservative to be captivated by superior storage methods. Adam has attended most of the annual conferences since I began attending in 2004, and told me that he would like to become a Governor of the Society. Not once did Adam ask me what work I do, and he evidently does not know because he was surprised when I told him I am not a Member of the Society for Cryobiology. Whether or not I am formally accepted as a Member, my attendance at these conferences is implanting me into the consciousness of the cryobiologists as being a member of their community.

Annotated bibliography of cryoprotectant toxicity

08. January 2012 · Comments Off · Categories: Cryonics, Science · Tags: Brian Wowk, Cryobiology, Cryoprotectant Toxicity, DMSO, Ethylene Glycol, Glycerol, Gregory Fahy, Vitrification

Introduction

Cryoprotectant toxicity should be distinguished from other mechanisms of cryopreservation injury such as chilling injury (injury produced by too low temperatures as such) and cold shock (injury produced by rapid cooling). Cryoprotectant toxicity itself can again be divided into general cryoprotectant toxicity and specific cryoprotectant toxicity. General cryoprotectant toxicity involves concentration (water substitution) effects of cryoprotectants and specific cryoprotectant toxicity involves the effects of individual compounds on cellular viability. General cryoprotectant toxicity presents a formidable obstacle for cryopreservation methods that require very high concentrations of cryoprotectant agents (such as vitrification).

Another mechanism of injury that is rarely discussed in the cryobiology literature but that can complicate cryopreservation of complex organs is “non-specific” dehydration injury. In light of the fact that the current generation of vitrification agents are delivered in hypertonic carrier solutions and contain non-penatrating cryoprotective agents which do not cross the blood brain barrier, this form of damage may be especially important in cryopreservation of the brain.

Systemic reviews of cryoprotectant toxicity are rare but some mechanisms for (specific) cryoprotectant toxicity have been proposed including, but not limited to, protein denaturation, modification of biomolecules, membrane injury, destabilization of the cytoskeleton, oxidative damage, and ATP depletion. It is important to stress that some of the mechanisms may be downstream effects of other mechanisms. For example, ATP depletion can cause oxidative damage. And as Gregory Fahy has pointed out, cryoprotectant toxicity should be distinguished from injury associated with the method of introduction and washout of the cryoprotectant. In 2004, Fahy, Wowk et al., proposed a compositional variable to predict general cryoprotectant toxicity.

Cryoprotectant toxicity can also vary by species and organ type. Cryoprotectants that are moderately toxic in one species can be highly toxic in others. Similarly, cryoprotectants that are moderately toxic in one organ can be highly toxic in others (or even between different types of cells within organs). This raises the question of whether universal non-toxic cryoprotective agents are attainable (a requirement for reversible vitrification in complex organisms).

Cryoprotectant toxicty can be investigated by cryopreserving an organ (or cell) and measuring its viability after rewarming and washout of the cryoprotective agent. To eliminate the influence of other mechanisms of injury associated with cryopreservation (such as ice formation), a cell can just be loaded and unloaded with the cryoprotectant without cryopreservation. The effects of hypothermia on viability can be eliminated altogether by normothermic perfusion of the organ. This, of course, introduces a challenge for hypoxia sensitive organs such as the heart and the brain because cryoprotective agents may not be good oxygen carriers.

Papers

Baxter SJ, Lathe GH (1971). Biochemical effects of kidney of exposure to high concentrations of dimethyl sulphoxide.
Biochemical Pharmacology. Jun; 20(6): 1079-91.

Baxter and Lathe investigated the effect of high concentrations of DMSO on kidney preparations. In a series of illuminating experiments, the investigators established that anaerobic glycolysis was reduced in slices and homogenates as a result of increased activation of the gluconeogenesis enzyme Fructose 1,6-diphosphatase (FDPase). DMSO-induced activation of FDPase can be inhibited by adding an amide or lysine to DMSO. The finding that a combination of DMSO and an amide allows for less toxic cryoprotectants formed the basis of subsequent investigations of GM Fahy for potent vitrification solutions.

Fahy GM (1983). Cryoprotectant Toxicity Neutralizers Reduce Freezing Damage.
Cryo-Letters 4: 309-314.

In this paper GM Fahy reports the ability of toxicity neutralizers urea, formamide, and acetamide (all amides) to reduce injury of cryopreserved renal cortical slices with DMSO. In later research papers Fahy will establish that DMSO neutralizes the toxicity of formamide, and not the other way around.

Fahy GM (1984). Cryoprotectant toxicity: biochemical or osmotic?
Cryo-Letters 5: 79-90.

If osmotic stress is an important cause of injury during introduction and removal of cryoprotectant agents, improved viability can be obtained by reducing the rate of cryoprotective agent introduction and removal. Fahy reviews the literature and presents data obtained in renal cortical slices that indicate that substantial hypertonic osmotic stress does not produce major changes in viability. Conversely, reducing exposure time to higher concentrations of the cryoprotectant can contribute to improved viability. These results suggest that biochemical toxicity, not osmotic stress, is the major factor in cryoprotectant-induced injury.

Fahy GM (1984). Cryoprotectant toxicity: specific or non-specific?
Cryo-Letters 5: 287-294

Fahy reviews the argument (Morris, Cryoletters 4, 339-340, 1983) that the lower toxity of cryoprotectant solutions that contain DMSO and amides can be entirely explained by the lower absolute concentration of DMSO. Fahy points out that the original Bexter and Lathe experiments demonstrated that solutions with the same absolute amount of DMSO (4.6 M) but with or without amides had different effects on glucose utilization. The author also presents data showing that “simple substitution (“dilution”) of one agent for another strikingly fails to reduce overall toxicity over a very critical range of DMSO concentration.” Also briefly discussed is the possibility of mutual toxicity neutralization between DMSO and amides, a topic that would be further explored by Fahy in future research.

Fahy GM, MacFarlane DR, Angell CA, Meryman HT (1984). Vitrification as an approach to cryopreservation.
Cryobiology. Aug ; 21(4): 407-26.

In this paper on vitrification as an alternative to conventional cryoprotection, Fahy et al., list a number of methods for reducing cryoprotectant toxicity:

Primary (direct) methods:

Maintain temperature as low as possible;
Select an appropriate carrier solution;
Keep exposure time at higher concentrations to a minimum;
When possible, employ specific cryoprotectant toxicity neutralizers.

Secondary (indirect) methods:

Avoid osmotic injury;
Mutual dilution of cryoprotectants may be helpful in some instances;
Use extracellular cryoprotectant to reduce exposure to intracellular cryoprotectant when possible.

The most important insights, some of which are still maintained in the current generation of vitrification solutions, concern toxicity neutralization, the choice of an appropriate carrier solution, and the use of extracellular cryoprotectants.

Fahy GM (1986). The relevance of cryoprotectant “toxicity” to cryobiology.
Cryobiology. Feb; 23(1) :1-13.

Fahy presents evidence that cryoprotectants themselves can present a source of injury. As a consequence, the advantages of higher concentrations of the cryoprotective agents does not necessarily produce higher viability after freezing, even when this allows for greater ice inhibition. He reviews data on “cryoprotectant-associated freezing injury” for DMSO, ethylene glycol, methanol, ethanol, and glycerol. Because vitrification requires very high concentrations of cryoprotective agents, toxicity is the key limiting factor in reversible vitrification of organs.

Fahy GM, Lilley TH, Linsdell H, Douglas MS, Meryman HT (1990). Cryoprotectant toxicity and cryoprotectant toxicity reduction: in search of molecular mechanisms.
Cryobiology. Jun; 27(3): 247-68.

Fah,y et al., delineate 6 criteria that must all be met simultaneously in order for a putative mechanism of cryoprotectant toxicity to be implicated:

The relationship between observed biochemical alteration and cellular viability must be clear or easily plausible;
The maginitude of the cryoprotectant effect must be large enough to be significant;
The effect must be irreversible over a reasonable time span after removal of the cryoprotectant;
The time course of the observed effect must be consistent with the time course of observed injury;
The cryoprotectant effect must be possible under conditions that could reasonably be encountered inside a living cell being prepared for freezing or being subjected to freezing and thawing itself;
The cryoprotectant effect must be due to the cryoprotectant itself and not due to the technique of introduction and washout.

The authors investigate the proposed mechanisms for the biochemical effects of DMSO toxicity in the 1971 Baxter study and find that a) the effect of DMSO on FDPase activation is too small to affect the normal respiration of the cell and therefore fails to meet criterion 2 to be a significant mechanism of cryoprotectant toxicity; b) the presence of formamide does not affect the interaction between DMSO and lysine; and c) toxicity is not consistently reduced by blocking alteration of FDPase rather than substituting those compounds for DMSO.

The authors further present results that do not support the theory that generalized protein denaturation is related to cryoprotectant toxicity. The article ends with a referenced list of phenomena possibly related to mechanisms of cryoprotectant toxicity.

Fahy GM, da Mouta C, Tsonev L, Khirabadi BS, Mehl P, Meryman HT (1995). Cellular injury associated with organ cryopreservation: Chemical toxicity and cooling injury.
Editors: John J. Lemasters, Constance Oliver. Cell Biology of Trauma, CRC Press

Fahy, et al., review different mechanisms of cryoprotectant toxicity with a particular focus on DMSO-medicated chemical injury. Mechanisms discussed include fructose-1,6-bisphosphatase activation, sulfhydryl oxidation, activation of extracellular proteinases and endothelial cell detachment and death. The article lists a number of interventions that do not change CPA-medicated injury such as inhibition calcium mediated injury or protein denaturation. The authors also report how the toxicity of formamide can be completely reversed by addition of DMSO.

Bakaltcheva IB, Odeyale CO, Spargo BJ (1996). Effects of alkanols, alkanediols and glycerol on red blood cell shape and hemolysis.
Biochimica et Biophysica Acta. 1280: 73-80

In this elegant and thoughtful paper, the authors use the human red blood cell to study cryoprotectant toxicity. Morphological observations, quantification of hemolysis, measurements of the dielectric constant of the incubation medium (Ds) and the dielectric constant of the erythrocyte membrane in the presence of organic solutes (Dm), are used to investigate cryoprotectant toxicity in a series of alkanols, alkanediols, and glycerol. The authors propose that toxicity of a cryoprotectant is related to its ability to change the ratio of Ds/Dm. Changes in this ratio reflect changes in the difference between hydrophobicity of the solution and the membrane, with decreases in this ratio leading to increased exposure of membrane surface area and vesiculation, and increases in this ratio leading to decreased exposure of membrane surface area and cell fusion. The authors suggest that the design of less toxic cryoprotective agents should involve the maintenance of dielectric homeostasis of the medium and the membrane. Their findings also throw light on the observation that combinations of various cryoprotectant agents (such as DMSO and formamide) can reduce the overall toxicity of a solution.

Fahy GM, Wowk B, Wu J, Paynter S (2004). Improved vitrification solutions based on the predictability of vitrification solution toxicity.
Cryobiology. Feb; 48(1): 22-35.

This seminal paper on non-specific cryoprotectant toxicity represents a major contribution to the cryobiology literature in general, and enabled the authors to formulate less toxic vitrification solutions for the cryopreservation of whole organs. In the paper the authors propose a new compositional variable that reflects the strength of water-cryoprotectant hydrogen bonding called qv*. Contrary to the cryobiology wisdom to date, the authors found that weaker glass formers favor higher viability. As a consequence, vitrification agents with higher concentrations of cryoprotective agents are not necessarily more toxic. Although qv* is not helpful in predicting specific cryoprotectant toxicity, this paper, and the research that is reflected in it, suggests that non-specific cryoprotectant toxicity is mediated through the effects of penetrating cryoprotectant agents on the hydration of biomolecules.

The 2011 Cryobiology Conference

11. August 2011 · Comments Off · Categories: Cryonics, Science · Tags: Arthur Rowe, Chilling Injury, Cryobiology, John Crowe, Microtubules, Peter Mazur, Society for Cryobiology Conference

July 24-27 I attended the 2011 annual Society for Cryobiology conference in Corvallis, Oregon.

A number of the first presentations were concerned with means to *avoid* cryopreservation. Room temperature storage is much less expensive and troublesome, and improves ease of transport, especially in remote areas. One such technology “shrink wrapped” DNA in a glass and another used trehalose to protect lipid membranes in a similar manner. Applied to cells, such technologies are viewed as a form of room-temperature vitrification.

Another researcher had successfully freeze-dried hematopoietic stem cells using trehalose and other additives without losing the ability of the stem cells to differentiate. Stress proteins in combination with trehalose allowed for desiccation of mammalian embryonic kidney cells without loss of viability. Late Embryogenesis Abundant (LEA) proteins also assist trehalose in dehydration tolerance.

Christoph Stoll showed that depleting red blood cell membranes of cholesterol can increase
trehalose uptake, but when I asked him in person about it, he said that the uptake was not enough to make much difference. Depleting cell membranes of cholesterol makes them more vulnerable to chilling injury, so I don’t think cholesterol depletion is a very good idea.

Masakazu Matsumoto spoke about some of the interesting anomalous properties of water.

Andrew Brooks spoke about the largest University cell and DNA repository in the world at Rutgers University. They store DNA by plunging in liquid nitrogen. He told me that 10 freezings and thawings does not impair DNA quality. That is encouraging for CI’s tissue/DNA storage program, because we plunge our samples into liquid nitrogen. Brooks gave data showing that RNA is much less hardy in liquid nitrogen than DNA.

David Denlinger noted that HSP70 RNAi can block cold tolerance in insects. He also mentioned a Czech study which found that insect larva fed proline could survive liquid nitrogen. Perhaps we should be feeding proline to terminal cryonics patients.

In preparation for this conference, I had done a lot of reading on the subject of chilling injury and was hoping to question researchers on the subject. Steve Mullen showed a video of meiotic spindles dissociating at low temperature.

Spindles are a form of microtubules. Microtubules are known to dissociate at low temperature, but can spontaneously re-associate upon rewarming. But that would not be so beneficial when the microtubules are functioning as centrosomes because the reassembly would not be a reconstruction of the original structure. This is probably why cell division often stops at low temperature.

Tiantian Zhang is one of the two candidates to become the new Society for Cryobiology President. Her field of study is cryopreservation of fish embryos and oocytes, which are especially vulnerable to chilling injury.

Fish are useful scientific models because they have a much simpler genome than mammals. 50% of endangered species are fish, but fish don’t get anywhere near the concern that pandas do. In both her lecture, and when I spoke to her in person, Dr. Zhang had apparently not learned any more than what was in her 2009 paper.

Why does reducing yolk content reduce chilling injury? Why is methanol the most non-toxic cryoprotectant for fish embryos, and so protective? If microtubule dissociation were a mechanism of chilling injury, it is indeed ironic that a 2006 Society of Cryobiology meeting presentation found that methanol causes proteolysis.

Kevin Brockbank spoke on the oxygenated hypothermic machine perfusion that he used to preserve pig livers at 4-6deg C for 12 hours. As a somewhat off-the-wall question, I asked him if he had assayed for chilling injury. This was off-the-wall because I have never heard of anyone assaying chilling injury. He responded that he had not, but that there were plans to use gene arrays to assay for chilling injury. This is like gene arrays to assay for aging — it requires deeper analysis, especially if chilling injury — like aging — is due to multiple mechanisms, the mechanisms are controversial, and no one mechanism is dominant. Northern wood frogs, arctic insects, and polar fish don’t have problems with chilling injury, although their adaptations include heat shock proteins and highly unsaturated cell membranes.

Much to my frustration, I have not had a good conversation with Peter Mazur (the uncrowned guru of cryobiology) since he got me to tell him I am a cryonicist several years ago. I have repeatedly asked him questions, and he has repeatedly been rude and dismissive. This year was different, for some reason. When I asked him about frozen water expansion contributing to mechanical damage he noted that cells could tolerate a 9% expansion without lysis even if freezing was intracellular. When I asked him how much dehydration cells could tolerate without damage, he said cells could lose all of the osmotic water (90% of cell water), and could lose more in freeze-drying with proper protectants (like trehalose). I was somewhat stunned by this answer, which takes no account of intracellular electolyte concentration increasing on dehydration. Next year I will be more optimistic about the possibility of talking with him, and I will prepare questions more carefully.

I spoke to Society for Cryobiology President John Crowe about his negative remarks concerning trehalose, in light of the fact that he is very aware of many of its benefits. John told me that a new method of manufacturing trehalose from starch is making trehalose as inexpensive as sucrose. If trehalose is used on bakery sugar, the sugar will not melt and run after a couple of days, as happens with sucrose. I mentioned to John that Robert Ettinger had just died. I had imagined that he might ask me to say a few words about the matter to the cryobiologists at their business meeting, but John treated the matter as a non-event, and I got the distinct impression that he would have preferred that I had not mentioned it.

At the business meeting it was noted that membership has dropped from close to 300 in 2008 and 2009 to just above 200 in 2011. There is concern that web access to the journal
CRYOBIOLOGY is becoming so easy that the incentives for membership have dropped. Or the global financial crisis is taking its toll on Society for Cryobiology membership. CRYOBIOLOGY journal impact factor has fallen to 1.830 from a high of 2.044 in 2002.

I appreciate being able to attend the business meetings, but one of the vehemently anti-cryonics cryobiologists gives me dirty looks. I have not been kicked-out yet, though, and decreasingly worry that I will be. A similar thought goes through my head as when I attend an Alcor meeting: “Spy in the House of Love.” But I really want the Society to prosper and grow, not be harmed, because I appreciate their good work (as with Alcor), even if they view me as a threat.

I had a brief chat with the cryonics-friendly Treasurer, who asked me when I think a cryonics patient will be reanimated. When I told him not less than 50 years, he said that a lot of surprising things can happen in 20 years. He is a more optimistic cryonicist than I am! At least as remarkable is that he is currently working with biotechnologists who are engineering scaffolds that can be used for growing organs from stem cells. That is a very cryonics-relevant project!

Every year I exchange a few words with Arthur Rowe (the cryobiologist who repeatedly compares cryonics to restoring a cow from hamburger — as he did in “Death in the Deep Freeze” – a comparison which probably originated with Peter Mazur). This year Arthur spent a lot of time hanging out with John G. Baust (the man who compared publishing cryonics science research with publishing Nazi hypothermia experiments). At the end of the conference I lost patience trying to catch Arthur alone, so I approached Arthur to say “hi”. Arthur said that he had seen on TV that Robert Ettinger had just died. He asked me about Robert’s educational credentials, and about my taking Robert’s place as CI President. Then he introduced me to John Baust. John was politely quiet, and said very little.

As with the 2010 Cryobiology Conference, I felt decreasingly paranoid as the meeting proceeded, but my level of paranoia was nonetheless very high near the beginning of this meeting. Overall, the amount by which I “came out” as a cryonicist was modest this year, and my softening of the hostility of cryobiologists to cryonics was modest this year compared to the previous one. The 2012 Society for Cryobiology Conference is scheduled to be held in Argentina.

The Future of Aging: Pathways to Human Life Extension

28. February 2011 · Comments Off · Categories: Science · Tags: Biogerontology, Gregory Fahy, Interventive Gerontology, Joshua Mitteldorf, Michael R. Rose, Michael Rae, Nanotechnology, NENS, Ray Kurzweil, Rejuvenation, Robert Freitas, SENS, Stephen Cole, The Future of Aging: Pathways to Human Life Extension, Thomas Donaldson

This book review was originally published in Cryonics magazine, 1st Quarter, 2011.

Editor-in-chief, cryobiologist, and aging researcher Gregory M. Fahy and his associate editors Michael D. West, L. Stephen Cole and Steven B. Harris have compiled what might be the most impressive collection of articles on interventive gerontology to date in their 866 page collection The Future of Aging: Pathways to Human Life Extension. The book is divided into 2 parts. The first part includes general, scientific, social and philosophical perspectives on life extension. The second part is a collection of proposed interventions, which are organized in chronological order, starting with the (projected) earliest interventions first. Of course, such an organization of the materials necessitates a subjective estimation of when such technologies will be available and is bound to be controversial. The collection closes with a number of appendices about contemporary anti-aging funding and projects (SENS, Manhattan Beach Project).

I have read the book with the following two questions in mind:

1. Which approaches for increasing the maximum life span show clear near-term potential?

2. Is meaningful rejuvenation possible without advanced cell repair technologies?

What follows are my comments on selected chapters of the book.

I cannot say that I am a big fan of Ray Kurzweil’s work. His general introduction to life extension, “Bridges to Life,” co-written with Terry Grossman, starts out on a restrained note, discussing the benefits of caloric restriction, exercise, basic supplementation, and predictive genomics. But it then ratchets up into bold claims about the future that rest on controversial premises: about biology and health following the same path as information technology; about the technical feasibility of molecular nanotechnology; and about the nature of mind. One thing that remains a mystery to me is how such an accelerating pace of anti-aging technologies could be validated considering the relatively long life expectancy of humans. Presumably we are expected to adopt a lot of these technologies based on their theoretical merits, success in animal studies, or short-term effects in humans.

Associate Editor Stephen Cole contributes a chapter on the ethical basis for using human embryonic stem cells. I suspect that his argument in favor of these therapies relies on adopting a definition of personhood that has more far-reaching, and more controversial, consequences than just permitting the use of human embryonic stem cells. One of the most disconcerting aspects of the bioethical debate on stem cell research is that many of its advocates seem to feel that if they do not see an ethical case against it, government funding for such research should be permitted. In essence, this means that opponents of embryonic stem cell research are obliged to financially support it as well. This is a recipe for further aggravating what has already become a passionate political debate.

As someone with relatively limited exposure to the biogerontology literature I should be cautious in singling out one technical contribution for high praise, but Joshua Mitteldorf’s chapter on the evolutionary origins of aging is one of the best and most inspiring articles in the field of aging research I have read and worth the hefty price of the book alone. Mitteldorf outlines a case for the theory that evolution has selected aging for its own sake and presents experimental findings that falsify other explanations for aging such as wear-and-tear and metabolic trade-offs. That aging is firmly under genetic control may appear the most pessimistic finding in terms of the prospects of halting aging but in fact allows for the manipulation of a number of selected upstream interventions that can inhibit or mitigate these programs.

It is clear from this ambitious book that cryobiologist Greg Fahy also has a strong interest in biogerontology but nothing prepared me for the encyclopedic knowledge that he displays in his lengthy chapter on the precedents for the biological control of aging. Fahy’s chapter further corroborates the view that aging is under genetic control. He also reviews a great number of beneficial mutations and interventions in animals and humans that can extend lifespan. Reading all these inspiring examples, however, I found myself faced with the same kind of despair as when reading about all the neuroprotective interventions in stroke and cardiac arrest. There is great uncertainty how such interventions would fare in humans (or other animals) and, more specific to the objective of human life extension, how we ourselves can ascertain that there are no long-term adverse consequences. Fahy does not run away from the most formidable challenge of all, rejuvenation of the brain without losing identity-critical information, but points out that identity-critical information might be retained despite the turnover and replacement of components that a meaningful life extension program for the brain would most likely require. Fortunately, people who make cryonics arrangements can feel a little better about this issue because their survival is not dependent on safe technologies becoming available in their lifetime.

Zheng Sui’s report on using high potency granulocytes to cure cancer in mice is one of the more exciting chapters in the book and a fine example of the role of chance discoveries in biomedical research (Zheng by accident discovered a mouse innately resistant to cancer). With substantial support of the Life Extension Foundation and other private donors, Sui is aggressively pursuing Leukocyte Infusion Therapy (LIFT) human trials instead of pursuing the torturous path of trying to illuminate the biochemical and molecular mechanisms that drive the successful results in mice. I should mention that a unique concern for cryonicists is that eliminating cancer in the absence of other effective anti-aging technologies could increase the likelihood of dying as result of identity-threatening insults such as cardio-vascular complications, ischemic stroke, or Alzheimer’s disease.

I must admit being somewhat disappointed in the chapter about “evolutionary nutrigenomics” by Michael Rose and his collaborators. Michael Rose has always struck me as one of the more level-headed and empirical aging researchers, and his work with fruit flies is a resounding demonstration of using evolutionary tools to investigate and combat aging. His short contribution to this book reads more as a quickly thrown together status update of their company, Genescient, than a rigorous treatment of the issues. Dispersed throughout the text are a number of interesting perspectives on alternative approaches to aging research and the validation of anti-aging interventions, but these issues are not discussed in much detail. Michael Rose’s work is of great interest, but this chapter is neither a good introduction to his work nor an in-depth treatment of the practical applications of his research.

Anthony Atala’s chapter, “Life Extension by Tissue and Organ Replacement,” is a fascinating update on the current status and potential of regenerative medicine and tissue engineering. Unlike most of the chapters in this book, the author reports a number of examples of successful clinical applications. It is a good example of how working with nature (instead of trying to improve upon it) can have meaningful near-term benefits. Unfortunately, there is no discussion of the progress in regenerative medicine for the brain. Obviously, such strategies cannot involve a simple replacement of the brain with a newly grown brain but selected repair technologies can play an important role in brain-damaging diseases and insults. The inclusion of “life extension” in the chapter title seems somewhat artificial to me because there is no distinct treatment about how tissue and organ replacement will be expected to contribute to life extension. Additionally, there is little discussion of contemporary artificial and mechanical alternatives to organs (or biological structural components) in this chapter, or in any other chapters in the book, which I think is a minor oversight.

Robert J. Shmookler Reis and Joan E. McEwen contribute a chapter about identifying genes that can extend longevity. Their discussion of the prospects for mammals includes the sobering observation that “many of the gains we can attain by a single mutation in the simpler organism may already have been incorporated in the course of achieving our present longevities.” Then again, unless aging is firmly under genetic control in simple organisms but the result of wear and tear in humans there should be (unique) approaches in humans that should confer similar benefits as well.

The publication of this book came to my attention when I learned about Robert Freitas’s contribution, “Comprehensive Nanorobotic Control of Human Morbidity (PDF),” so I was quite interested in reading this final chapter of the book. I am not qualified to comment on the technical aspects of his vision of nanotechnology. I think it is fair to say, though, that if resuscitation of cryonics patients is possible they will most likely be resuscitated in a future that has nanomedical capabilities resembling those that are outlined in this chapter. For this reason alone, this chapter should be of great interest to readers of this magazine. Of particular interest is the discussion of cell repair technologies and brain rejuvenation, a topic of great interest to cryonics. Freitas devotes considerable space discussing how anti-aging strategies like SENS can be achieved with medical nanorobots but the chapter falls short of offering a distinct exposition of a nanomedical approach to aging and rejuvenation. With such profound molecular capabilities one would think that such an approach would not just consist of updating existing biotechnological approaches to eliminate aging related damage with more powerful tools. I think that the distinct capabilities that molecular technologies have to offer would have benefitted from a more extensive discussion of their transformative capabilities. In particular, the section on nanorobot-medicated rejuvenation could have benefitted from a more rigorous treatment of the question of how these interventions would produce actual rejuvenation. Rejuvenation will be a practical requirement for most cryonics patients and it would be interesting to see a more detailed technical discussion of this topic.

Robert Freitas introduces the phrase NENS (Nanomedically Engineered Negligible Senescence) for his vision of how the goals of SENS can be achieved through nanomedicine. This raises an important question: is there any reason to believe that the timeline for “conventional” SENS will be different from the timeline for mature molecular medicine? It is hard to tell, but one could argue that the development of mature nanotechnology is more comprehensive than any strategies designed to deal with the causes or effects of the aging process. So why not just fund the work of biological and mechanical molecular nanotechnologists to accelerate meaningful re-design of the human organism? I think that the best answer is that our current state of knowledge does not justify giving a privileged position to any particular approach and having these visions of the future compete may be the best hope that we have for seeing meaningful rejuvenation and the resuscitation of cryonics patients in the future.

If there is one serious omission in this impressive collection of articles it is a more comprehensive chapter on the topic of biomarkers of aging in humans. As reiterated throughout this review, the gold standard and most rigorous determination of the efficacy of anti-aging therapies and interventions is to empirically determine whether they increase maximum human lifespan. For obvious reasons, most medical professionals and healthcare consumers are pressed to make decisions based on less rigorous criteria and the development of a set of reliable biomarkers of aging is highly desirable. Of course, the most rigorous case for successful biomarkers would require the same kind of long-term studies, leading to an infinite regress problem. How to break out of this predicament while retaining a framework to make rational decisions about life extension technologies is not a trivial problem and can be the topic of a whole new volume of articles. Interestingly enough, one of the most insightful perspectives on this issue is given in Appendix A by SENS researcher Michael Rae when he points out that therapies aimed at rejuvenation can be tested at much more rapid timescales than therapies to retard the aging process or increase the maximum lifespan.

Michael Rae also notes that SENS’s “engineering heuristic” is well established in other fields of biomedicine. It is certainly the case that aging research could benefit from a stronger emphasis on solving problems and repairing damage instead of completely trying to understand the underlying pathologies but it also needs to be pointed out that the engineering approach has not fared much better in areas of research that are notoriously resistant to effective solutions such as neuroprotection in stroke. Ultimately, the SENS approach cannot completely escape studying the mechanisms and metabolic pathways involved when treatments are compared and side-effects are studied. In this sense, the difference between SENS and alternative approaches is a matter of degree, not principle.

I think that the editors are justified in claiming that the prospects for solving the aging challenge have never looked better. A close inspection of all the chapters, however, shows that no significant interventions in the aging process in humans are available now, and I doubt they will become available in the near future. And even if the aging process can be eliminated, there will still be medical conditions and accidents that require placing a person in cryostasis until effective treatment is available. For the foreseeable future there is good reason to agree with Thomas Donaldson’s advice* that making cryonics arrangements is the most fundamental and sensible decision one can make in order to reap the benefits of powerful future life extension therapies.

*Thomas Donaldson – Why Cryonics Will Probably Help You More Than Antiaging, Physical Immortality 2(4) 28-29 (4th Q 2004)

Philosophy of science and life extension

27. January 2011 · Comments Off · Categories: Science · Tags: Empiricism, Life Extension, Logical Positivism, Metaphysics, Paul Edwards

Paul Edwards concludes his chapter ‘The Semantic Challenge’ in his book God and the Philosophers with the following observation about logical positivism:

It is not uncommon nowadays to hear logical positivism dismissed as a set of crude errors and confusions. This is done with an air condescension by philosophers whose writings are usually models of obscurity. To people of my generation who came to philosophy in the 1940s, when traditional metaphysicians were a dominating force, logical positivism was a liberating movement. Occasionally the leading figures were guilty of dogmatism, and on some important issues, such as the mind body problem and the question of free will, the logical positivists made no significant contributions, but the main doctrines seem to me substantially sound. The verification principle in particular, when stated with suitable amendments, is a powerful weapon against pretentious humbug.

Do life extensionists need to take an interest in philosophy of science and metaphysics? In his review of James Ladyman and Don Ross’s Every Thing Must Go: Metaphysics Naturalized, Alcor staff member Mike Perry notes that “as immortalists we hope to be in the world for a good long while, thus we are interested in the nature of reality. Reality determines, among other things, what our prospects are for our own longterm survival.”

Alternatively, one could argue that metaphysics is not a theoretically legitimate discipline and that the verifiable claims of physics exhaust what we can say about “reality.” Perhaps the most useful benefit of familiarizing oneself with philosophy of science and analytic philosophy is that it enables one to get a better appreciation of the difference between meaningful experimental science and sweeping generalizations deduced from shaky metaphysics.

Further reading: Five important empiricist philosophy books

The RhinoChill: A New Way to Cool the Brain Quickly

20. January 2011 · Comments Off · Categories: Neuroscience, Science · Tags: Cooling, Cryonics, Hypothermia, Ischemia, Mike Darwin, Neuroprotection, RhinoChill

We scientists are difficult, cranky, and above all, maddeningly frustrating people. Want to turn lead into gold? No problem, we can tell you how to do that, and in fact have even done it already: the only catch is that the cost of such ‘nuclear transmutation’ is many times that of even the most expensive mined gold. You say you want to travel to the moon? Done! That will be ~$80 billion (in 2005 US dollars). Want to increase average life expectancy from ~45 to ~80 years? Your wish is our command, but be mindful, you will, on average, spend the last few of those years as a fleshpot in the sunroom garden of an extended care facility.

And so it has been with an effective treatment for cerebral ischemia-reperfusion injury following cardiac arrest. Thirty years ago, laboratory scientists found a way to ameliorate most (and in many cases all) of the damage that would result from ~15 minutes of cardiac arrest, and what’s more, it was simple! All that is required is that the brain be cooled just 3^oC within 15 minutes of the restoration of circulation. The catch? Well, this is surprisingly difficult thing to do because the brain is connected to the body and requires its support in order to survive. And the body, as it turns out, represents an enormous heat sink from which it is very difficult to remove the necessary amount of heat in such short time. Thus, the solution exists and has been proven in the laboratory, but it has been impossible to implement clinically. This may be about to change as a variety of different cooling technologies, such as cold intravenous saline and external cooling of the head begin to be applied in concert with each other. Separately, they cannot achieve the required 3^oC of cooling, but when added together they may allow for such cooling in a way that is both effective and practical to apply in the field. A newly developed modality that cools the brain via the nasal cavity may provide the technological edge required to achieve the -3^oC philosopher’s stone of cerebroprotection.

Read the complete article in PDF here.

Institute for Evidence Based Cryonics

Resuscitation and Reintegration of Cryonics Patients Symposium

In praise of cold

Preserving and inferring

ApoE4, diet, and gender

The 2012 Society for Cryobiology Meeting

Annotated bibliography of cryoprotectant toxicity

The 2011 Cryobiology Conference

The Future of Aging: Pathways to Human Life Extension

Philosophy of science and life extension

The RhinoChill: A New Way to Cool the Brain Quickly

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