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:

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

Secondary (indirect) methods:

  1. Avoid osmotic injury;
  2. Mutual dilution of cryoprotectants may be helpful in some instances;
  3. 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:

  1. The relationship between observed biochemical alteration and cellular viability must be clear or easily plausible;
  2. The maginitude of the cryoprotectant effect must be large enough to be significant;
  3. The effect must be irreversible over a reasonable time span after removal of the cryoprotectant;
  4. The time course of the observed effect must be consistent with the time course of observed injury;
  5. 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;
  6. 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.

On October 27-29 I attended CR VII, the 2011 Calorie Restriction Society Conference held in Las Vegas, Nevada.

Members of the Calorie Restriction Society restrict their calories while maintaining adequate nutrition as a means of extending their lifespan (or improving their healthspan), as has been proven to work in lower animals.

Although I was still in a wheelchair as a result of falling from a ladder and hip surgery, I got my airline to give me handicapped-support (wheelchair assistance), and I rented a wheelchair in Las Vegas.

CR VII was the seventh CR Society conference held in the ten years since the first such conference was held in the same city, in the same hotel, and in the same meeting-room ten years earlier in 2001. Thursday, October 27 featured presentations by Calorie Restriction Society Members, whereas Friday and Saturday featured presentations by PhD scientific researchers. I am a CR Society Member, so I was invited to speak on cryonics on Thursday. It was a small conference, so there were not many more than forty people attending on any of the days.

My presentation was preceded by a presentation by Peter Voss, who is both a CR Society Member and a Member of Alcor. Peter and his companion Louise Gold were the only CR Society Members other than me attending  the conference who are cryonicists. Peter spoke of the ultimate goal of indefinite lifespan, sharing his wisdom based on his experience practicing calorie restriction, describing cryonics as a “safety net of unknown fabric”, and mostly speaking of his goal of developing Artificial General Intelligence to accelerate research in life extension technologies. Concerning his CR practice, he noted that CR is not binary, and that people receive the benefits to the degree that they restrict their calories. He said that he does not count calories, but simply weighs himself and adjusts his calories appropriately, which is the practice I have adopted. Peter is not worried about hostile AIs because he believes that rationality is positively associated with morality. (See http://www.adaptiveai.com/ for a sample of Peter’s work.)

Although it was not a large group, I expected that such a group of dedicated life extensionists willing to go to extremes in restricting their calories would be very receptive to the practice of cryonics. On the other hand, Shannon Vyff warned me that although CR Society Members can be enthusiastic to hear about cryonics, they don’t sign-up. I gave considerable thought to the marketing aspect of my presentation. I decided to be very up-front about being a salesman, while nonetheless attempting to side-step salesmanship (and sales resistance) by concentrating on the technical issues and encouraging a technical discussion (although I did mention prices and insurance funding).

Alcor Member (and long-time cryonics promoter) Brenda Peters lives in Las Vegas, so I invited her to be my guest at the CR Society Conference. My thought was simply that Brenda and I could renew our friendship while enjoying the conference together.

I began my presentation by describing my and experience and mistakes in practicing calorie restriction as well as my fall in September which resulted in hip surgery and no prospect of walking again for many weeks — and how this had interrupted by exercise/CRAN program. When I asked who felt familiar with their knowledge of technical issues of cryonics, I was surprised that none of the non-cryonicists raised their hands.

After giving my presentation of the technical issues in cryonics I asked the audience to pair-up to discuss both their understanding of my presentation, and reasons they may have for thinking that cryonics may not work. After the paired discussions I asked for questions and objections. Brenda was more enthusiastic than I expected about raising her hand to comment. I somewhat bluntly said that I would rather hear from anyone but her, which was apparently confusing to people who weren’t aware that we knew each other. I was wanting to hear the unvarnished objections to the idea of cryonics which CR Society Members might have. I did not mean to hurt Brenda’s feelings, and I blame myself for not discussing my expectations with her beforehand. I did, nonetheless, allow Brenda to speak a couple of times.

It proved to be hard work getting CR Society Members to explain whatever objections they might have to cryonics. One fellow expressed his belief that not enough is known about the mind to know that cryonics can preserve it. I replied that the mind is based on the synaptic “connectome” and that minds recover from low-temperature surgery in which there is no electrical activity in the brain. Another fellow wanted to hear the experimental evidence that cryonics patients have been revived, to which I could only reply that cryonics is dependent on technologies which do not yet exist, and that revival seems inevitable to me if technology continues to progress and the anatomical basis of mind is preserved. One man believed that dogs had already been cryopreserved and revived, but I corrected his misconception by stating that the dogs have only been revived from cooling down to just above the melting temperature of water. When someone said that most businesses don’t last long, I replied that it is a mistake to compare the durability of cryonics organizations to efforts to start a diner in a location where the success is uncertain. One woman raised the overpopulation issue, which I noted is no more a plausible threat than the danger that too many people will practice Calorie Restriction. I added that the same logic would ban all medical research, especially research into preventing infectious diseases.

Although there were not many objections, neither did I hear much enthusiasm for cryonics. Perhaps they were stunned by an unfamiliar idea, and it takes time for resistance to be overcome. I had been hoping for some sign-ups. I had placed Membership forms on the literature table. It was as if they had no objections to cryonics, but still weren’t interested. Which left me thinking that I shouldn’t have asked for reasons why they think cryonics won’t work, but instead asked for reasons why they won’t sign-up.

A number of people complimented me on the quality of my presentation. But during subsequent discussions with CR Society Members at the conference, I heard further objections to cryonics. One CR Society Member told me that he hoped my presentation would motivate him to sign-up for cryonics. He said that he had mentioned cryonics to his mother several years ago, but she was freaked-out by the thought of being reanimated in a strange and alien world. Since then she had become demented, and he thought it would be wrong to foist cryonics upon her while she is in that condition.

Another CR Society presenter spoke of his project to develop an eco-friendly farm with local barter and community-building that would be sustainable through the disastrous global warming and prolonged depression he was expecting. His bleak vision of the future of technology left no possibility for cryonics, but at least he corrected himself when he started to say “cryogenics”.

Another fellow I spoke with later was concerned that cryonics organizations could not survive in light of the acrimony he saw between Members. His biggest concern, however, was that people of the future would be vastly superior, and treat him with contempt or worse upon his revival. A female CR Society Member told me that she is restricting calories entirely to increase her health-span, not her lifespan. She does not think that life is very good, and she has the hope and belief that the afterlife will be better.

Over lunch, one fellow suggested promoting cryonics as a means of cutting the astronomical health-care costs that so many people incur in their last year of life. I replied that any association of euthanasia with cryonics or any hastening of death on the expectation that cryonics may work would be disastrous for cryonics — and all the moreso if done as a cost-cutting measure.

I had difficulty moving around in the conference room due to the tables and my wheelchair, which made it difficult to chat with people during breaks. I had a similar problem during meal breaks. Whether I would have gotten a better understanding of why no-one seemed eager to sign-up for cryonics if my mobility had been better remains to be seen. I would think that after years of giving presentations about cryonics I would become blunted to lack of interest, but each such experience remains uniquely poignant and disappointing.

I learned much from the scientific presentations, but I won’t attempt to summarize very much. I was, however, very impressed by the extent to which a linkage was made between the blockage of the insulin/IGF-1 pathways in lower organisms and the practice of calorie restriction by humans. There is evidence that protein restriction may be the essence of calorie restriction, and that low protein diets are associated with reduced levels of IGF-1, but only when protein is less than 12% of macronutrients. Increasing insulin sensitivity seems to be the key to extending lifespan, yet although exercise is the most powerful intervention increasing insulin sensitivity, exercise does not increase lifespan.

Stephen Spindler and Luigi Fontana are scientists who have a long and intimate relationship with the CR Society. Both were speakers at this conference. Luigi in particular has been conducting studies on the physiology of long-time calorie restriction practitioners, and the benefits that are seen in the risk factors for various aging-associated diseases. He has published many studies of this research:

http://www.ncbi.nlm.nih.gov/pubmed/21402069

http://www.ncbi.nlm.nih.gov/pubmed/21841020

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2724865/

http://ajpheart.physiology.org/content/294/3/H1174.long

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2829867/

A DVD of the presentations is being made by the CR Society, and will be available for sale within a few weeks, I expect.

August 31 to September 4, 2011 I attended fifth biannual SENS Conference (SENS5, Strategies for Engineered Negligible Senescence) at Cambridge University in the United Kingdom.

People who attend SENS conferences are the demographic that is the most receptive to cryonics of any identifiable group I have yet found. They are mostly scientists interested in intervening in the aging process. Quite a number of attendees are already cryonicists, including Aubrey de Grey, the originator of SENS and the organizer of the conference. But cryonicists are nonetheless a distinct minority. In previous years I brought a few Cryonics Institute brochures, which were soon taken. This year I brought enough brochures for as many of the 240 attendees as might want one (there were many left over).  I also brought a few copies of my “Scientific Justification of Cryonics Practice” (the published write-up of my SENS3 cryonics presentation) which I gave to a few attendees who seemed most receptive.

In addition to my oral presentation on cryonics I also had a poster. Scientific conferences usually have poster sessions where scientists present research, reviews, or ideas in the form of a poster. Poster presenters stand by their posters at scheduled times to discuss their work on a one-to-one basis with individuals rather than to an audience. My poster dealt with challenging the concept of biological age and denying the possibility of a biomarker of aging that could determine biological age. I contended that biological age and biomarkers of aging assume a singular underlying aging process, which I denied on the grounds that aging is multiple forms of damage. I sought to make maximum use of the one-to-one interaction by preparing Socratic questions to stimulate thinking and discussion with the attendees. The process also gave me another means of meeting and speaking to those attending. One interesting person I met was a Torontonian who is currently studying for his PhD at University of Glasgow. His work involves developing gene vectors that can precisely target and modify genes on chromosomes. I consider gene therapy to be an essential tool for the ultimate implementation of SENS, and a deficiency of SENS that there is so little attention paid to this technology. I don’t see how SENS can be implemented by any means other than genetic re-programming. LysoSENS, for example, would require new genes to create new, more effective enzymes for the lysosomes. MitoSENS would require all mitochondrial proteins be made in the nucleus and imported into the mitochondria.

Partly in this connection, was my aggressive lobbying of Aubrey de Grey to have Argentinian biogerontologist and Cryonics Institute member Rodolfo Goya as an invited speaker at SENS5. I began lobbying in January when Dr. de Grey was at ConFusion 2011. Aubrey was initially reluctant based on the first batch of Dr. Goya’s papers that I sent, but a later batch in which Dr. Goya was principle investigator proved to be effective. In Dr. Goya’s presentation at SENS5 he described his use of viral vectors attached to magnetic nanoparticles to deliver IGF-1 genes to senescent female rats to rejuvenate dopamine-producing cells in the hypothalamus. He injects the particles into the venticles, so the technique is somewhat invasive. Another speaker, Matthew Wood, described exosome nanoparticles which can cross the blood-brain barrier so I am hopeful that Dr. Goya can adopt this technique. Dr. Goya ended his presentation with a short pitch for cryonics (showing CI’s cryostats), which even I found embarrassingly awkward. I introduced Dr. Goya to a number of other cryonicists attending SENS5, including Igor Artyuhov, who is the scientific advisor for KrioRus, and Alcor Member Maria Entraigues, who is the SENS volunteer co-ordinator, and a native of Argentina (now living in Los Angeles).

Russian biogerontologist Alexey Moskalev reported on decreasing the number of single-strand DNA breaks and increasing the maximum lifespan in fruit flies by overexpressing the stress response/DNA repair gene GADD45 in the nervous system. That such a presentation would be included in SENS5 was of special interest to me insofar as I have contended that (and debated with Aubrey de Grey concerning) nuclear DNA damage possibly being a significant cause of aging damage that is missing from SENS:

http://www.benbest.com/lifeext/Nuclear_DNA_in_Aging.pdf

http://www.alcor.org/magazine/2011/02/28/deficiencies-in-the-sens-approach-to-rejuvenation/

http://www.alcor.org/magazine/2011/06/07/sens-a-reply-to-ben-best/

Alexey later told me that he had read my paper in REJUVENATION RESEARCH, and I’d like to think that I helped inspire his work.

Alexey announced that there will be a genetics of aging conference in Moscow in April 2012. I entertained the thought of going, partly because of my desire to see KrioRus, but I would rather go later when KrioRus is established in its new building, and has a research program in full swing.

Alexey’s research was partly funded by the Science for Life Foundation (the organization of the wealthy life-extensionist Russian Mikhail Batin). Maria Konovalenko (who was featured in LONG LIFE magazine) reported on her work at the Science for Life Foundation to build an open web-based database of age-related changes (molecular and phenotypic). Maria has her own blog.

I am not going to attempt to describe the other very excellent SENS5 presentations other than to say that great progress has been made in starting research programs on each of the SENS strategies, and by 2012 research on all the strategies is expected to be in progress.

Alcor President Max More was an invited speaker, which means that he had a half-hour time-slot immediately preceding my 15-minute time-slot near the end of the program. Max gave an overview of cryonics, whereas I concentrated on technical and scientific issues associated with vascular and neuronal injury from ischemia and reperfusion. During the question period I was asked if we are interacting with hospital staff to limit pre-mortem ischemia in cryonics patients. I said that the current legal environment limits such interactions, but that pre-mortem anti-oxidant protocol has been recommended and used.

I arranged to send more information to a few people in the audience, including a man who was interested in hydrogen sulfide to limit ischemic injury in cryonics, and an Italian neuroscientist who is interested in neurophysiology studies of vitrified brain tissue as well as contact information for Italian cryonicists.

At the final banquet I sat with CI Member Dr. Gunther Kletetschka, who is now living in the Czech Republic and is pursuing a number of imaginative cryonics-related research projects. One of these involves carbon nanotubes to deliver non-toxic metals to cells to use magnetocaloric cooling. Such a technique could cool tissues uniformly rather than externally, thereby eliminate the thermal stress that causes cracking when vitrified cryonics patients are cooled at cryogenic temperatures.

The last day was spent punting on the Cam River, with dinner in the evening. This provided an opportunity for more networking and information exchange, although most of this was in connection with biogerontology.

There was much biogerontology to be learned at SENS5. What I learned at SENS5 can potentially extend my life and that of others. To postpone cryopreservation by life extension is to benefit from technical advances, to extend the time in which I can contribute to technical advancement, and to enjoy more present life. In the best case, rejuvenation will become a reality in my lifetime and I won’t need to be cryopreserved at all. I work for this possibility as well as for improved cryopreservation. Moreover, in doing research for my cryonics presentation at SENS5 — and in giving the presentation — I learned many things that can help me make more informed choices in directing the research that Aschwin and Chana de Wolf do for the Cryonics Institute.

A video of my presentation may eventually be placed on the SENS5 YouTube site.

Many people in the life extension community follow some kind of diet. Historically, caloric restriction (CR) has been the most popular and most discussed option. Other popular diets include the Mediterranean diet and the Paleolithic diet.  In one sense, comparing these diets is like comparing apples and pears. The emphasis of caloric restriction is on how much we eat (given adequate nutrition) and the other diets are more concerned with what we eat. People who follow certain diets may also have different aims. In the case of CR, life extension. In the case of the Mediterranean diet, preventing and delaying cardiovascular and neurodegenerative diseases. And many who adopt a low-carb diet are (initially) motivated by securing sustainable weight loss.

Assuming that diet plays some role in longevity and disease, it is rather obvious that cryonicists should take a strong interest in choosing the right diet. As it looks to me, there are a number of important considerations.

1. The most important aim of a diet for cryonicists should be to avoid, or delay, neurodegenerative diseases. Extending your life and ending up with advanced Alzheimer’s Disease is worse than dying young and being cryopreserved under circumstances that optimize preservation of personal identity.

2. The choice to follow a particular diet should work for your genotype. Admittedly, nutrigenetics is a very young field but there is a growing recognition that human evolution has not stopped since the start of agriculture and that different populations respond differently to certain diets. And even within these populations we should expect individuals to respond differently to diet.

3. A decision to follow a certain diet should be based on empirical evidence, not on intuition, abstract theories, or thought experiments. In the case of choosing diets, this  means identifying a diet that has shown a favorable ratio of good outcomes in experimental studies, and humans in particular.

Putting this all together, it seems to me that a low calorie diet remains the most defensible choice for most cryonicists because it has been studied longer, studied more extensively, and has the most robust favorable outcomes. CR also seems to stand out favorably in that there are relatively few studies that find detrimental outcomes and its benefits seem to embrace many species and populations. Another advantage of CR is that it can capture all the important goals that life extentionists seeks from a diet: longevity, weight loss and prevention (or delay) of neurodegenerative diseases.

It may be the case that many of the benefits of CR actually come from a reduction of carbohydrates. But one of the problems with a paleolithic diet is that it may be more beneficial for certain populations than others. As Gregory Cochran and Henry Harpending demonstrate in their seminal book The 10,000 Year Explosion: How Civilization Accelerated Human Evolution, human evolution did not stop when hunter gatherers started agriculture, and some populations are more adapted to agricultural products (such as milk) than others. Another concern about the paleolithic diet is the controversy surrounding saturated fat. For life extentionists who carry one or two copies of the ApoE4 gene, a diet high in saturated fat may actually increase the probability of Alzheimer’s disease. Others dispute this and recommend a diet high in (saturated) fat to prevent dementia.  In light of this uncertainty, the most prudent course of action may be to incorporate the emerging evidence against carbohydrates into a CR diet without emphasizing saturated fat.

There is an ongoing debate whether the longevity benefits of CR will be as great in humans as in lower species but the evidence so far seems to be that there are at least benefits in terms of delaying the onset of age-associated diseases. Whether these benefits are conferred through a change in gene expression or because they reduce the amount of chemicals that can participate in pathological events is not clear, but our incomplete knowledge about the mechanisms involved should not deter anyone from following CR. As I currently see it, the role of ongoing research into nutrigenetics and other diets should be to further calibrate and refine a low calorie diet to optimize it for a specific individual and to further delay the onset of neurodegenerative diseases.

CR seems to come closer to being a universal diet than other diets but it may be contra-indicated for some people, such as certain athletes and extreme ectomorphs. There are also cases in the life extension community of people who pushed it too hard (or neglected good nutrition), offsetting all the gains from the diet, or even endangering their own health. A diet that does not make a person feel good, is generally not a diet that is good, let alone one that can be sustained over time.  The aim of a diet should not be to conform to an impersonal set of recommendations, but to monitor your own response and increase the chance for personal survival.

According to Steve Jobs, death is such a great benefit to mankind that it would have to be invented if it did not exist:

No one wants to die. Even people who want to go to heaven don’t want to die to get there. And yet death is the destination we all share. No one has ever escaped it. And that is as it should be, because Death is very likely the single best invention of Life. It is Life’s change agent. It clears out the old to make way for the new. Right now the new is you, but someday not too long from now, you will gradually become the old and be cleared away. Sorry to be so dramatic, but it is quite true.

As the baby boomers age, we can be sure to hear a lot more of what the cryonicist Mark Plus has called, ‘Humanist Death Apologetics.’ Never mind the horror, the destruction, and the suffering that comes with death, because, “it clears out the old to make way for the new.” Fortunately, a more enlightening perspective on death has been offered by the philosopher Herbert Marcuse:

It is remarkable to what extent the notion of death as not only biological but ontological necessity has permeated Western philosophy–remarkable because the overcoming and mastery of mere natural necessity has otherwise been regarded as the distinction of human existence and endeavor…

A brute biological fact, permeated with pain, horror, and despair, is transformed into an existential privilege. From the beginning to the end, philosophy has exhibited this strange masochism–and sadism, for the exaltation of one’s own death involved the exaltation of the death of others…

Modern market economies demonstrate on a daily basis that death is not necessary for the old to make way for the new. Neither do people have to be faced with death to have a meaningful life. Steve Jobs invites us not to be “trapped by dogma” but, unfortunately, he embraced the biggest dogma of all; the idea that human mortality is a good thing and gives meaning to life.

The reader is encouraged to explore some alternative views about death and aging:

Robert Freitas Jr – Death is an Outrage

Ben Best – Why Life Extension?

Aubrey de Grey – Old People Are People Too: Why It Is Our Duty to Fight Aging to the Death

26. September 2011 · Comments Off · Categories: Cryonics, Health · Tags: , , , ,

As every modern consumer knows, smartphones are today’s go-to portable technology. Everything from GPS navigation to finding a good deal on your next meal or haircut right NOW to a wide variety of games and applications may be had at the touch of a button. But developers of smartphone applications (i.e, “apps”) are only just beginning to realize the true capabilities of having so much computing power in the palm of your hand. Indeed, the possibilities for health monitoring applications in combination with GPS location bodes well for cryonicists.

Until cryonics-specific apps become available, there are several existing applications useful to cryonics members and organizations. Here are some of the most interesting from the Android Market:

ICE (In Case of Emergency):   Emergency personnel look for ICE information in patient mobile phones. This ICE app has a couple of widget options and can be accessed even when the phone is locked. My favorite feature is the ability to put any special instructions (like the protocol from your Alcor bracelet) on the main screen. The app acts primarily as an emergency contact list. Your cryonics service provider should be #1, followed by family and friends who support your cryonics arrangements. Additionally, you may enter your vital stats, medical and dental insurance information, and any known allergies, conditions, and/or medications.

For those with “dumb phones,” just create a contact called “ICE” and enter your cryonics organization’s emergency number. Additional information about placing ICE  numbers in your cell phone may be found in this article by Fred and Linda Chamberlain.

Emergency Button: Emergency Button sends a distress signal with your coordinates to a defined recipient when pressed. This has obvious utility for all matters of personal safety, and can be used to alert your cryonics organization to emergency health situations as soon as they emerge.

Google Latitude: Latitude is a GPS location tracking app. It allows for various privacy settings and can be configured to share only with specific people. A cryonics organization could, with its members’ permission, use such an app for real-time location tracking.

These are just three basic apps that are commonly available and useful to cryonicists now. I hope to be updating this list as improvements in smartphone technology continue.

Personalized Cryonics is an approach to cryonics that emphasizes the use of individual (health) information to optimize a person’s cryopreservation circumstances and outcomes.

To exchange information and empower individuals, a moderated discussion list was created by the Institute for Evidence Based Cryonics. It is a discussion list for members of existing cryonics organizations who seek to understand and change their personal circumstances to optimize their own survival and (potential) cryopreservation.

Typical topics on this list include personal genomics, personalized medicine, diet options, fitness, nutrigenetics, cryonics first-aid, custom-built stabilization equipment, advance directives and living wills, third-party interference, brain threatening diseases, and local support groups.

The recent issue of Cryonics magazine features a comprehensive update on intermediate temperature storage (ITS). This article contains an important observation:

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.  Higher fracturing temperatures are believed to occur when tissue freezes instead of vitrifies because freezing increases the glass transition temperature of solution between ice crystals.  The temperature at which fractures begin is therefore believed to be a surrogate measure of goodness of cryoprotection, with lower temperatures being better.

This is an important observation because one of the arguments that has been made against intermediate temperature storage is that Alcor routinely records fracturing events above the nominal glass transition temperature (Tg) of the vitrification solution. But if we recognize that such events can be (partly) attributed to ice formation due to ischemia-induced perfusion impairment it should be obvious that the recording of fracturing events above Tg as such cannot be an argument against ITS. After all, we also do not argue against the use of vitrification solutions because ice formation will still occur in ischemic patients that are perfused with vitrification solutions. Because cryonics patients almost invariably suffer some degree of ischemia prior to cryoprotective perfusion and cryopreservation, our knowledge about fracturing events in “ideal” human cases remains incomplete.

But even if ITS would only be successful in reducing fracturing events, instead of completely eliminating them, this should not be an argument against ITS. To argue that a technology should not be used because it does not completely eliminate a problem would constitute a sharp departure from the philosophy that has informed Alcor since its formation. In many areas, the evolution of Alcor’s technologies has been one of incremental evidence-based progress towards better procedures and storage conditions, not one of radical change.

The worst argument against ITS is that mature repair technologies will be able to repair clean fractures. It is a poor argument because one could similarly argue that advanced cell repair technologies will also be able to reverse the biochemical effects of short periods of ischemia and moderate degrees of ice formation. What distinguishes Alcor from other cryonics organizations is that it aims to secure viability of the brain as far into its procedures as it practically can. In ideal cases, this currently means meeting the challenge of further reducing cryoprotectant toxicity during cryoprotectant perfusion and reducing/ eliminating fracturing.

Perhaps the biggest obstacle to offering ITS to the general Alcor membership is cost. An obvious solution would be to offer ITS in addition to conventional liquid nitrogen storage. An alternative would be to gradually phase out conventional liquid nitrogen storage by no longer offering it to new neuro members and to raise cryopreservation minimums accordingly. The (preliminary) cost estimates in the article indicate that this would bring the cost of ITS for neuros closer to that of conventional liquid nitrogen whole body cryopreservation. The article does not provide specific information on the “greater capital costs” of whole body ITS systems but the reported lower liquid nitrogen consumption per patient for whole body systems suggests that it might be possible to offer whole body ITS without putting it beyond the reach of most (new) members with adequate funding.

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.

Reportedly, when James Watson and Steven Pinker had their genome sequenced, they declined to know their risk for Alzheimer’s disease. Clearly this is not an option for life extensionists and cryonicists, who are better off knowing whether they have a copy or, worse, two copies of the ApoE4 gene.

Patri Friedman, son of the libertarian economist David Friedman (who in turn is the son of the Nobel laureate Milton Friedman), recently learned that he has two copies of the ApoE4 gene when 23andMe updated their reports. Caucasian and Japanese carriers of two E4 alleles have between 10 and 30 times the risk of developing Alzheimer’s by 75 years of age, as compared to those not carrying any E4 alleles. Patri is a life extensionist, practitioner of the paleo diet, and recently made cryonics arrangements with his whole family at Alcor – and is thus far more prone to a pro-active course of action.

When he realized that there was no good central resource for people with copies of the ApoE4 gene he started a new blog called ApoE4 – The Ancestral Allele, which aims to share practical information and research for health-conscious E4 carriers. The first posts discuss some of the benefits of having the E4 gene (better episodic memory) and what kind of diet is recommended for E4 carriers. He also encourages guest posts and other co-bloggers to help run the website.