The Brain Preservation Technology Prize: A Challenge to Cryonicists, a Challenge to Scientists

[FEATURED ARTICLE]

Cryonics, 2nd Quarter 2011

For an illustrated version of this article, click here.

For a response to this article by Alcor staff member Mike Perry, click here.

By Kenneth J. Hayworth, Ph.D.

My name is Kenneth Hayworth and I am a PhD neuroscientist working in a university laboratory developing automated electron imaging techniques. The primary focus of my research is tracing synaptic connections in brain tissue at the ultrastructure level. I am also a long-time, albeit quite skeptical, member of Alcor.

Like many of my fellow materialist scientists I have no problem viewing the idea of cryonics as merely a technical challenge: “Can a dying person be placed in a long-term static state to await future technology that can revive and cure them?”   As a neuroscientist I have no problem stating the minimum conditions that such a static state needs to meet for it to allow possible future revival of the individual with memories and personality intact – the precise connectivity of the brain’s hundred billion neurons must remain intact. I have discussed the idea of cryonics with dozens of my fellow neuroscientists over the years and this is the central question that comes up again and again:

“Do current cryonic suspension techniques preserve the precise wiring of the brain’s neurons?”

The prevailing assumption among my colleagues is that current techniques do not. It is for this reason my colleagues reject cryonics as a legitimate medical practice. Their assumption is based mostly upon media hearsay from a few vocal cryobiologists with an axe to grind against cryonics. To try to get a real answer to this question I searched the available literature and interviewed cryonics researchers and practitioners. What I found was a few papers showing selected electron micrographs of distorted but recognizable neural tissue (for example, Darwin et al. 1995, Lemler et al. 2004). Although these reports are far more promising than most scientists would expect, they are still far from convincing to me and my colleagues in neuroscience.

It is often assumed that the only evidence that will persuade large numbers of mainstream scientists to embrace cryonics is a demonstrated revival of a whole mammal after being cooled to a temperature sufficient for long-term storage (an extremely difficult technical goal which is likely still decades off). Such a demonstration of revival might be the only acceptable criterion for the small cryobiology community (who is used to thinking of the brain as a ‘black box’ which either survives or does not), but this is not necessarily a criterion neuro and cognitive scientists have. For these brain science specialists, who probably outnumber cryobiologists a hundred to one, the key criterion is a demonstration that the precise connectivity of the brain’s 100 billion neurons is preserved by cryonic procedures.

To reemphasize, I believe that thousands of neuro and cognitive scientists are ready and willing to embrace cryopreservation as a legitimate medical procedure if it can be shown that cryonic procedures preserve the precise pattern of connectivity between neurons across the entire brain. What’s more, according to the top cryonics researchers I have interviewed, the current techniques may be up to this task (e.g. Lemler et al. 2004).

The action item to the cryonics community should be clear: Today’s best available imaging technology should be used to rigorously determine the quality of neuronal circuit preservation within a cryopreserved brain, and the results should be widely publicized so that every mainstream scientist has an opportunity to see the true current state of cryopreservation for him or herself.

Brain Preservation Technology Prize

After considerable thought I came to the conclusion that the best way to bring about such a wide-reaching ‘scientific reevaluation of cryonics’ is to put forward a challenge prize modeled after the inspirational Ansari X Prize (for commercial space travel) and the skeptical Paranormal Challenge Prize offered by the James Randi Educational Foundation.  A prize has the crucial advantage of precisely defining the criteria for success – a fact all cryonics skeptics should eagerly embrace. Simultaneously a prize has the ability to explain to a wide audience why the particular milestone chosen (in this case demonstration of brain preservation at the electron microscope level) is on the critical path to a truly inspirational future goal (reanimation of a preserved individual). Put simply, a challenge prize will bring skeptics, advocates, scientists, and interested laypeople to the same table for an impartial evaluation of cryopreservation and a thoughtful conversation about what we can reasonably expect to achieve over the next few years.

As a neuroscientist whose day job is to map neural circuits, I know exactly what type of evidence is needed to convince the scientific community that cryonics preserves the neural circuits encoding our unique memories and personality. What is required is a systematic whole-brain survey with an electron microscope. Recently I, along with my colleagues John Smart and Jacob DiMare, formed the Brain Preservation Foundation (BPF) to promote new scientific research in the field of whole brain preservation for long-term static storage. The BPF has announced the Brain Preservation Technology Prize (purse currently at $106,000) for the first team to demonstrate that an entire large mammalian brain can be preserved for long-term storage such that the connectivity between neurons remains intact and traceable using today’s electron microscopic imaging techniques. A complete set of rules for the prize can be found on our BPF website www.brainpreservation.org.

A challenge to cryonicists – demonstrate the quality of your product

This prize is being presented as a challenge to cryonics providers like Alcor and their research partners: “Demonstrate the quality of your product in a rigorous, independent, and open way to the scientific community and to your customers.” The BPF is hard at work raising funds to promote this prize and to help perform the electron microscopic evaluation required, and we are recruiting a board of scientific advisors and judges that will give the prize credibility. This prize should be viewed as a tremendous opportunity for the cryonics community as a whole to publicly refute the prevailing negative stereotype of frostbitten and destroyed brain tissue. Even if the current cryonic techniques are unable to meet the rigorous requirements for winning the prize, a ‘good showing’ should serve to reinvigorate interest in cryonics in the mainstream scientific community and in the general public as well. It is my fervent hope that Alcor and its research partners will rise to this challenge. As a long-time dues paying member of Alcor, I believe it is Alcor’s responsibility to do so to counter the continual claims in the press that their service is inadequate.

A challenge to scientists – develop alternatives to cryonics

The Brain Preservation Technology Prize is also a challenge to the wider scientific community. It has been almost 50 years since the professional cryobiology community briefly considered the possibility of putting a person into indefinite suspended animation for medical applications and then quickly dismissed the possibility as impossible with the technology of the day.  Incredible advances have been made in all areas of science and technology in the intervening decades. Is it still impossible to preserve a person in a long-term static state? If so, why? The Brain Preservation Technology Prize is a challenge to this generation of scientists to reevaluate what is possible, to move beyond the expectations of their parents and grandparents and look at the problem with a fresh perspective.

We at the BPF believe that one crucial part of this fresh perspective is to consider true alternatives to cryopreservation including room temperature chemical fixation and plastic embedding of the brain. Such a ‘chemopreservation’ approach, which has exactly the same goal as cryonics (i.e. placing a dying person in a long-term static state to await future technology that can revive them) was suggested decades ago (Olson 1988) but it has never been seriously pursued. In chemopreservation, fixatives like glutaraldehyde and osmium tetroxide are used to physically bind the molecular components in tissues together preventing decay reactions from occurring even at room temperature. Following application of fixatives, a solvent-based dehydration process is used to remove all of the water within the tissue and replace it with a liquid polymer which can then be cured (Hayat 2000). The result is a hard plastic block containing a piece of brain tissue in which all the water has been removed from every nook and cranny of intra and extracellular space and has been replaced with hardened plastic. The structure of the original neural circuits is perfectly preserved in this plastic matrix creating, in essence, a perfect fossil which preserves every synaptic connection in great detail in a completely inert state that can remain for centuries unchanged even at room temperature.

This chemopreservation process is routinely used in laboratories around the world to preserve small pieces of brain tissue (typically less than one cubic millimeter in volume) for study under the electron microscope. In fact much of what we know about the fine structure of neurons and synapses is owed to this chemopreservation process. Below is an electron micrograph of a piece of mouse brain tissue that was preserved by this standard method in my laboratory. A single synaptic connection is shown highlighted in color and reconstructed in 3D.

This nanoscopic regime of synaptic connectivity is the level at which our unique memories, skills, and personality traits are written. The target of the Brain Preservation Technology Prize is to demonstrate a surgical technique (cryo, chemo, or any other) capable of preserving an entire human brain with this level of fidelity.

Can this standard chemopreservation technique be applied to an entire human brain? Not without modification. Current protocols call for simply immersing the small pieces of tissue in the chemical fixatives. Slow diffusion of these chemicals puts a strict limit on the volume of tissue that can be preserved by such an immersion technique; however, if the technique is adapted to instead perfuse these chemicals directly through the brain’s vascular network an entire brain should be able to be preserved with the same fidelity. I have written a review (available on the BPF website) of the existing literature on such whole brain chemical perfusion; the review starts with experiments in the 1960’s showing that a whole brain can be perfusion fixed with osmium tetroxide (Palay 1962). I conclude in that review that whole brain chemical fixation and plastic embedding is absolutely possible with today’s technology, it is only a matter of refining the protocols. I know of at least two laboratories that are currently trying to develop these whole brain chemical fixation and plastic embedding protocols for the mouse, and several other researchers have contacted me (as a direct consequence of the Brain Preservation Technology Prize announcement) who are interested in developing these techniques for demonstration on a large mammal.

Putting an end to the ‘Cold War’

For the last forty years a very public war has been fought between the advocates of cryonics and professional cryobiologists. This is despite the fact that there has always been considerable overlap between these groups. In 1991 Mike Darwin wrote an excellent article entitled “COLD WAR: The Conflict Between Cryonicists and Cryobiologists” giving a detailed history of the origins of this conflict. He concluded that it had little to do with the science of cryopreservation and much more to do with a clash of ideals between a few prominent individuals. This clash however snowballed into an ugly drawn out war because of the perceived need of the cryobiologists to vigorously distance themselves from macabre media reporting of some of the earliest attempts at human cryopreservation. Instead of calling for a temporary halt to human cryopreservations so that a minimum acceptable protocol could be outlined, a few prominent cryobiologists instead went on the offensive claiming that any preservation attempt on a human was futile and should be banned. Cryonicists, indignant that their right to pursue a means of personal survival was being trampled, fought back and continued to perform amateur preservations that all parties today would agree were next to hopeless. Cryobiologists in turn began to purge cryonics advocates from their professional ranks and summarily reject their papers and grant applications. The ensuing decades have only entrenched this mutual animosity.

As Mike Darwin so aptly points out, this war has next to destroyed both sides in the conflict. The field of cryobiology, originally glamorized in the public eye as pursuing the goal of reversible suspended animation for emergency medicine and space travel (think of the movie 2001: A Space Odyssey), has withered – its public stand against cryonics was synonymous with a dampening of enthusiasm for these advanced applications. The practice of cryonics, although it has managed to advance significantly in the intervening years, has paid an even higher price. There might have been dozens of professional, well-funded research labs competing with each other over the previous decades to perfect the art of human cryopreservation. The public rejection by professional cryobiologists directly prevented this from happening. If things had been different we could reasonably expect that by now every hospital would have a professional cryopreservation team on call when needed, ready to perform a regulated emergency preservation procedure known to be of high quality. Instead we have only a few unregulated companies perpetually on the brink of bankruptcy offering cryopreservation services of unknown quality under legal circumstances that preclude optimal preservation.

It is time to put an end to this ‘Cold War’, and I believe the Brain Preservation Technology Prize is the perfect vehicle to do this. The traditional framing of the cryonics debate within the mainstream scientific community has always been set by the cryobiologists: “Until a person can be revived from cryonic suspension the entire practice should be viewed as quack medicine; after all, the person is already dead.” The Brain Preservation Technology Prize sidesteps this tired old debate by instead directly framing cryonics relative to the goals of the neuroscience community: “Can a cryonic (or other) preservation technique preserve the precise pattern of synaptic connectivity in the brain that is known by modern neuroscience to be the substrate for memory and individuality?”

The mainstream neuroscience community (much larger, more respected, and better funded than the cryobiology community ever was) retains high aspirations about its future success. Recent decades have seen tremendous strides in our theoretical understanding of the brain at the molecular, synaptic, neuronal, neural circuit, and systems levels. These advances in our theoretical understanding have been accompanied by an incredible sophistication in our ability to image the brain even at the ultimate level of tracing individual synaptic circuits. New automated electron imaging techniques (e.g. Denk & Horstmann 2004, Knott et al. 2008) have been invented within the last seven years which can image a chemically fixed and plastic embedded piece of brain tissue with nanometer resolution such that all synaptic connections between neurons within a small block can be determined with certainty. In fact, a recent paper in the journal Nature used one of these automated electron imaging techniques to reconstruct the precise neuron-to-neuron wiring of cells in a retina and compare that wiring diagram to recordings of the neurons’ functioning while alive (Briggman et al. 2011). Conclusion: the neurons’ original functions could be predicted based upon their traced connectivity to other neurons in the plastic block.

As a result of these technology developments, it is now quite acceptable among neuroscientists to discuss the future possibility of mapping an entire human brain at the synapse level (Kasthuri & Lichtman 2007) and the future possibility of simulating an entire brain (Markram 2006). In private discussions I have had with dozens of neuroscientists over the years, I have found that most readily agree that in the future we will create fully artificial brains that will be intelligent and conscious in a human way. Many agree that we will eventually be able to upload individual human minds into computers by scanning their brain circuitry.

Given their progressive attitude toward the future, I have no doubt that the target goal laid out in the Brain Preservation Technology Prize will be readily embraced by researchers in the neuro and cognitive sciences. Once the first teams begin to show real progress toward winning the prize, I fully expect to see a watershed change in attitude toward the idea of cryonics within the scientific community as a whole – at this point the ‘Cold War’ will be ended and a new era of cooperation between the scientific community and the cryonics community will have begun.

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Evaluation procedures for the Brain Preservation Technology Prize

The prize calls for a comprehensive statistical survey of the entire preserved brain at electron microscope resolution (~5 nanometers) to verify that the neuronal connectivity of the brain is preserved throughout. Because imaging an entire brain at such high resolution would be extremely costly and time consuming the prize only calls for imaging the brain at 1mm intervals and only calls for these slices to be statically surveyed at medium and high resolution looking for damage. The prize also calls for the extraction of three small sub volumes of the brain to be sectioned at 50nm thickness and serially imaged to produce a 3D volume image. Such 3D volume images are the only way to verify that synaptic connectivity is truly preserved by a given technique.

Evaluation of a chemopreserved brain embedded in a plastic block for long-term storage is straightforward – the block is milled down at 1mm intervals and the resulting surfaces are polished flat with a diamond knife and scanned by electron microscope. Evaluation of a cryopreserved brain is more challenging. One approach shown here is to warm the brain, wash out the cryoprotectant solutions, and then reperfuse the brain with fixative. The soft brain can then be sliced at 1mm intervals with a vibrating knife and each resulting slab put through a staining and plastic embedding procedure and SEM imaged. This warm-fix-embed approach has been used to evaluate the quality of cryopreserved brains previously. Another approach (which is technically more challenging) would involve leaving the cryopreserved brain in a vitrified state and electron imaging milled and polished surfaces directly.

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Kenneth Hayworth is president and co-founder of the Brain Preservation Foundation.  He is currently a postdoctoral researcher at Harvard University. Hayworth is co-inventor of the Tape-to-SEM process for high-throughput volume imaging of neural circuits at the nanometer scale and he designed and built several automated machines to implement this process. Hayworth received a PhD in Neuroscience from the University of Southern California for research into how the human visual system encodes spatial relations among objects.

References

Briggman, K. L., Helmstaedter, M., Denk, W. (2011). Wiring specificity in the direction-selectivity circuit of the mammalian retina. Nature, vol. 471 p183-188.

Darwin, M. (1991). “Cold War: The Conflict Between Cryonicists and Cryobiologists”. Cryonics, June 1991.

Darwin, M., Russell, S., Wakfer, P., Wood, L., Wood, C. (1995). Effect of Human Cryopreservation Protocol on the Ultrastructure of the Canine Brain. BPI Tech Brief 16, May 31, 1995.

Denk, W., Horstmann, H. (2004). Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS Biology, vol. 2(11): e329

Hayat, M.A. (2000). Principles and Techniques of Electron Microscopy Biological Applications. Fourth Edition. Cambridge University Press.

Kasthuri, N., Lichtman, .W. (2007). The rise of the ‘projectome’. Nature Methods, vol. 4 p307-308.

Knott, G., Marchman, H., Wall, D., Lich, B. (2008). Serial Section Scanning Electron Microscopy of Adult Brain Tissue Using Focused Ion Beam Milling. The Journal of Neuroscience, vol. 28 (12) p2959-2964.

Lemler, J., Harris, S.B., Platt, C., Huffman, T. (2004). The Arrest of Biological Time as a Bridge to Engineered Negligible Senescence.  Annals of the New York Academy of Sciences, vol. 1019 p559-563.

Markram, H. (2006). The Blue Brain Project. Nature Reviews Neuroscience. vol. 7, p153-160.

Olson, C.B. (1988). A Possible Cure for Death. Medical Hypothesis, vol. 26 p77-84.

Palay, S. L., McGee-Russell, S. M., Gordon, S., Grillo, M. A. (1962). Fixation of neural tissues for electron microscopy by perfusion with solutions of osmium tetroxide. Journal of Cell Biology, vol. 12 p385-410.