On 06 May, 2012 responses were solicited to what was termed The Cryonics Intelligence Test which was posted here on Chronosphere (see: http://wp.me/p1sGcr-vV). Two people responded to this public request to “take the test” and provide input on possible solutions to the problems posed by the resource material that accompanied the test. The test consisted of the resource materials and the following  instructions:

Dear ______,

If you can figure out the scientific take home message for cryonics in what is to follow, you will have demonstrated extraordinary insight into “thinking in a cryonics-medical context.”

You will also have the tool to be able to understand why I believe that cryonics must, on a purely scientific-medical basis, be pursued in a fundamentally different way, both biomedically and socially.

The Test: The test resource materials are available for download at ___________, you will find a number of full text peer reviewed scientific papers. In addition, you will be sent several cryopatient case Hxs. Together, these resources contain data which should give a reasonably intelligent person with a properly prepared mind a fundamentally new insight into a major, indeed overwhelming flaw in how cryonics has been, and currently is practiced.

Your task is to:

a) identify the problem(s)

b) identify one or more possible solutions

You have 5 days to complete this task. Your response should be in the form of a succinct statement of the problem, and an itemization, and if you like, a discussion of possible solutions.

Thanks for your patience and cooperation.

Mike Darwin

Purposes

The reasons for  this exercise were as follows (in no particular order):

To answer the question posed to me by Alcor CEO on what was the most important research to be undertaking in cryonics at this time.

To determine if a representative cross section of people not actively employed in cryonics, or working in cryonics-related research, would independently reach the same or same similar conclusions about a heretofore not understood or appreciated major problem in cryonics and propose the same possible solutions (or novel ones) to said problem.

To evaluate the caliber of the intellects (who chose to participate) who read Chronosphere.

To attempt to determine the number of Chronosphere readers who were willing to accept the challenge of  exposing their judgment and intellectual performance to scrutiny, either by myself, publicly, or both.

To determine the approximate number of people who took the time and exerted the effort to at least peruse the article and download the Test Resource Materials.

To attempt to get a preliminary idea of the nature of the readers of Chronosphere and their interest in highly technical topics of serious relevance to cryonics.

To gauge the impact and reaction of both the leadership of the cryonics community, and the cryonics community itself, to the revelations that result from this exercise and the commentary that is to shortly follow it.

To solicit novel solutions to the central problem posed in the exercise.

To inform the community at large, both the cryonics community and the public, of this serious problem in the way human cryoprerservation is currently being pursued (e.g., informed consent).

Participants

Two people (Alexander McLin and Gerald Monroe ) responded to the public request on Chronosphere to take the test. Prior to publicly soliciting responses, fifteen individuals of diverse backgrounds in cryonics were privately asked to take the test. Of these, eleven agreed to do so and of those eleven, ten completed the test. Of the ten privately solicited respondents, three agreed to allow publication of their answers; two with the use of their names. One individual, a young academic pursuing advanced graduate degrees, asked for and was granted anonymity, due to the likelihood that open involvement in cryonics could prejudice his academic career.

Since it is not possible for the responses of those who chose not to allow publication to be evaluated here, I will not make any comment on them beyond noting that they exist and that they, along with those of the respondents who did allow publication, were material in making the decision to pursue an open solicitation here for additional respondents.

At this time, the answers of the respondents are being presented absent any biographical/background information, so as not to bias the reader as he reads and considers each response. At a later date, I will edit this post to add a brief (few sentences) background description on each of the participants in order to provide demographic data on the participants as a group (e.g., how many were biomedically sophisticated, laypersons, long-time cryonicists, novices, etc.).[1]

 Responses

Responses are presented in alphabetical order (by name of the respondent). The only editing that has been done is to to correct typographical errors.

Alexander McLin

After studying the test materials, I have come to the following conclusions about how cryonics is currently practiced today and the problem with its current standards of practice. The problem is that cryonics isn’t effectively managing ischemia, nor it doesn’t seem to be incorporating medical findings about how the brain is affected by hypotension, hypoventilation, and hyperventilation.

Moreover, research in determining a method to predict onset of cardiac death after life-saving treatments is withdrawn indicate that this is difficult to do so, this in conjunction with other papers, show that the brain damage begins almost as soon as a patient’s circulatory system begins to fail. This is problematic from the cryonics point of view, because long before cardiac death is declared, the brain may have already suffered irreversible ischemia damage preventing optimal cryonics suspension.

The research materials furthermore show that hyperventilation when administrated for whatever reason actually makes things worse and that hypoventilation is preferred. With this in mind, do cryonics providers incorporate that finding when administrating oxygen to patients as part of the stabilization protocol?

To summarize, the conclusions I arrived at are that current cryonics providers are failing to manage ischemia, failing to research ways to predict the degree of severity of ischemia, failing to engage in proactive activities to minimize ischemia pre- and post-deanimation, and not incorporating medical findings in improving brain survivability in presence of hypotension and hypoventilation. In addition, there appear to be a lack of an attempt to maintain extensive database of patient medical history, collection of body fluids for pre and post-deanimation, and pre- and post-suspension which is essential for research intended to improve cryonics practices.

Here I will discuss solutions I have come up to address some of the conclusions I have arrived at. The biggest problem is the issue of ischemia and how likely it is to occur once oxygen is interrupted and also how sensitive the brain is to reperfusion injury. I would review the existing protocols to ensure whether they’re adequately taking the reperfusion injury into account, whether medicines need to be updated(add or remove medicines) with respect to the latest medical findings. It should be verified via meaningful actual research whether the cool-down equipment is really minimizing ischemia.

Finally, how can cryonics address the crucial issue of the existing medical-legal atmosphere that require patients to be declared dead according either to the cardiac or brain death definitions. Both which ensure that the brain will suffer ischemia damage before suspension occurs. How can cryonicists safely arrange for optimal cryonic suspension free of problematic legal implications? This suggests a need to engage in policy lobbying and pushing for legislation aimed towards changing the legal situation for the betterment of cryonics. To put it so bluntly, it appears that voluntary euthanasia is a cryonicist’s best friend, as distressing and stressful it may sound.

Lastly, cryonics providers need to establish a medical database and engage in much more data collection than they are doing at present. Some of the patient histories show recurrent problems with their collection equipment, do they need to be upgraded or replaced? Research in minimizing or preventing ischemia should be undertaken to determine how to optimize brain preservation prior to beginning suspension.

Mark Plus

Many cryonicists in hospice conditions currently deanimate and are pronounced after agonal periods similar to shock which result in prolonged hypoperfusion and hypoxia of the brain. These lead to significant compromises of the brain’s vasculature (e.g., the brain’s ability to self-regulate its blood flow to certain regions like the hypothalamus when the arterial pressure drops below 40 mm Hg) and interfere with cardiopulmonary support, washout and especially perfusion with cryoprotectants, not to mention the havoc they must cause to the brain’s fine structure.

Also, the trend towards harvesting organs from patients who are pronounced cardiac-dead after as little as two minutes of asystole is probably not a good thing for cryonicists, if the laws change to make it harder to opt out of such donations which will have the effect of ensuring thorough brain death.

My suggestions:

Use people with professional training in shock medicine and anesthesiology to perform the cardiopulmonary support after pronouncement. Monitor the level of brain perfusion with the proprietary bispectral index technology (which I had to look up and I’d like to read more about) to determine if brain hypoperfusion happens. Hypoventilate the patients.

Premedicate cryonicists before pronouncement with drugs like piracetam, arginine vasopressin and NO inhibitors, mentioned in the papers you sent me. You also wrote that Jerome White had attempted to premedicate himself with over the counter supplements until a few weeks before his suspension.

Cryonicists with terminal illnesses should consider moving to places where the laws allow assisted euthanasia so that they can go into arrest and undergo the suspension procedure well before their agonal decline.

Cryonics organizations need to gather a lot more data when they perform suspensions based on the current state of the medical art. The S-100B assay should be used along with other assays to measure brain injuries. These assays plus the bispectral index data can provide badly needed feedback on the effectiveness of brain perfusion procedures.

If the patient can’t deanimate at the time of his choosing, use some of the medical models developed by the DCD researchers to better estimate the patient’s time of cardiac death during standby.

I hope my answers and recommendations are not too off the mark, and I suspect I’ve misunderstand or failed to notice some key points. You gave me a lot of unfamiliar material to absorb in a short amount of time. After a few more weeks of study, I could probably understand it better. Some kind of primer would also help. A few years ago I speculated that based on actuarial considerations, the ideal candidate for cryosuspension would have to be a healthy ten year old who could walk into the lab and lie down on the table. That leaves the rest of us somewhere away from optimal candidacy for cryosuspension. But then, what can we do about it?

And I do plan to study this further, so thank you very much for the scientific background information, and feel free to send me additional papers.

Other observations:

I notice the contrast between the thorough reports you’ve written for the suspensions you’ve performed versus the ones written by Alcor’s “pod people,” which apparently includes Aaron Drake. Several things seem to go wrong with about every suspension Alcor has done lately, including basic preparations like not having the tray of all the necessary surgical tools ready for Dr. Nancy or the surgeon. I knew in a vague way that things had gotten bad, but you’ve given me some idea of how bad.

The scientific literature started to report the effects of shock and hypoperfusion decades ago, but you wouldn’t know that from the “official” cryonics propaganda. It seems like the cryonics movement should have incorporated this knowledge from the very beginning, but then physicians, surgeons and neuroscientists have mostly avoided cryonics and deprived us of their expertise. Dr. Ravin Jain, a neurologist, sits on Alcor’s board, and he should know this stuff, but I don’t get the impression that he’s done anything to incorporate his knowledge into Alcor’s suspension procedures. The neglect gives cryonics a reputation for “scienciness” and pseudoscience which it doesn’t necessarily have to have.

Gerald Monroe

a. The current techniques practiced for all the cryonics cases most likely result in long periods of ischemic hypoperfusion to the brain. Instruments now exist to detect this, combining the bispectral index with near infrared spectroscopy, and apparently even when top notch experts support cardiac surgeries on children, the hypoperfusion is common.

The ischemia and the hypoperfusion are very, very bad. Of course, so is the freezing. And the storage in liquid nitrogen where dissolved oxygen can reach the tissues and oxidize them. And the shoestring budget (compared to even a single hospital) the cryonics organizations have to do everything on.

b. It doesn’t sound like these problems are insoluble if there were real resources (compared to those spent to delay death from cancer by a few months, for instance) dedicated to the problem. Tomorrow, if cryonics had the resources of a single major metropolitan hospital, it could actually solve these problems in a systemic way.

There have to be experiments done on animals, where many different techniques* are attempted and evaluated. Evaluations should be done by preparing synapses of slices of the subject’s brain following the freezing. Also, rewarming and function tests (of slices), once the state of the art reaches the point that this is practical.

The human patients have to be part of this evaluation. If no one looks, the mistakes made will never be corrected. Somehow very small pieces need to be removed as samples from the human patients, following each cryonics procedure, small portions mostly taken from sections of the patient’s brain not thought to contain unique personality information.

And so on. Real improvements don’t come easily or cheaply – they come incrementally, with great effort, and honest evaluation of the results of each change. The last element is probably the most important of all.

The history of medicine is littered with many, many examples where something becomes common practice without honest testing of the results. Pretty much universally it fails.

With all that said, for those of us right here, alive in an era where cryonics does not have the resources it deserves, it is simply Pascal’s wager. No matter how dim the odds are, some chance of a form of survival is better than none. Information is probably duplicated inside the human brain many times over, and all of the decay processes that work against cryonics are things that happen according to predictable laws of physics. In a future world where a brain could be scanned at the molecular level, there is probably at least some recoverable memory and personality data for even the worst cryonics case.

For some, the prospective of saving even an incomplete fragment of yourself is better than the guaranteed destruction by rotting in the ground or burning in an incinerator.

Why it is like it is : the cryonics organizations don’t have any money. There’s probably a hundred new things that could be tried, and most of them are not better than what is being done now. Every dollar spent now is a buck less that could go to protecting the existing patients over many more decades.

Moreover, without any way to evaluate the current baseline : how effective is cryonics actually preserving the patients, right now? Making changes blindly is stupid. In the history of medicine, time and time again, it has been found that when a simple and dumb medical technique is compared honestly to a more expensive and advanced technique, almost universally the difference is minimal to none. A few examples : diuretics work as well as the far more expensive and specific beta blockers, film X-rays provide basically the same therapeutic improvement as the vastly more expensive CTs and MRIs, physical therapy works about as often as spine surgery, etc.

This is why in countries with socialized medicine, with outdated equipment and techniques and long wait lists, the patients live almost as long. (and the population lives years longer due to better lifestyles)

* A few ideas that might or might not work :
1. More rapid cooling by exposing the brain to coolant with burr holes and connecting pumps directly to cerebral perfusion
2. Drugs to prevent the cerebral arterioles from closing when exposed to cold perfusate.
3. Calcium blockers to prevent apoptotic pathways from triggering
4. Oscillating magnets like the Japanese claim work for transplanting teeth
5. Skipping cryonics entirely and plastinating the brain

Jordan Sparks, DMD

Well, I’ve read all the papers. I’ve attached the notes I made. I know you said I could skim them a little more quickly, but I was having trouble understanding and remembering. I needed to use a more aggressive approach this time. I did the references to help me get organized, and if I had to do that again I would do it without listing out all the names. Anyway, this is where I’m at.

I have a tentative answer which I may refine later. I’m continuing to think about it. You only gave me one cryopatient case Hx. I notice that it’s rich with hematology and chemistry data. Repeated samples were taken and charted over time. Both the TBW circuit and the cryoprotective perfusion circuit are well documented. Pressures and flow rates are nicely charted. Also, glycerol, blood gas, and pH were monitored during cryoprotective perfusion. The lab samples, in particular, are notable because that is not the current practice of Alcor or CI. It would take me some time to look back through case reports to see when was the last time this was done.

a) Cryonics providers are currently disregarding complexity associated with the biochemical milieu. I’m not quite sure how to state it, but all of the 22 papers treated their problems as a complex interplay of the mechanical issues as well as the biochemistry. Reading current Alcor and CI reports, on the other hand, there is a total disregard for the role of biochemistry.

That’s my first stab at it. I wish I could state it better, and I might try to rewrite it. I might wait for feedback from you before I go much further in case I’ve missed your point.

1.  Fast recovery from shock used vasopressor combined with hypertonic saline starch.  Slow recovery used fluid resuscitation.  Propofol and Hb concentrations were comparable in both groups.  The fast recovery resulted in better cerebral perfusion and a higher BIS that was likely due to the better perfusion.  CPP =MAP−ICP.

2.  Three resuscitation protocols: 1=FR (fluid resuscitation), 2=NA/HS (noradrenaline/ hypertonic starch), and 3=AVP/ HS (arginine vasopressin/HS).  The AVP/HS group had faster and higher increase in MAP and CCP as well as better survival.  Also, ICP was lower.

3.  After significant hypervolemia, cerebral circulation decompensation occured.  There were significant regional variations in cerebral blood flow.  The redistribution favored the areas related to cardiovascular control.

4.  Patients in shock can have normal physiological, hematological, fluid, and electrolyte balance but still die due to metabolic abnormalities.

5.  In spite of mechanisms for preferential shunting of blood to the brain, low MAP will result in poor perfusion.  This results in inadequate oxygenation as well as inadequate lactate washout.  Decreased perfusion leads to ischemic damage.

6.  Hemorrhagic hypotension was induced in dogs which was still above the lower limit of cerebral autoregulation.  This resulted in an increased turnover of free fatty acids in the CSF.

7.  Moderate reduction of MAP in anesthetized cats resulted in no significant EEG changes.  Below 40 mm Hg, cortical rhythms slowed and then stopped.  Cell damage was only found below 40 mm Hg.

8.  Baboons were pretreated with Phenoxybenzamine (PBZ) before hypovolemic shock, and it prevented the fall in cerebral blood flow.  EEG does not normally return after reinfusion.

9.  Bispectral index (BIS) dropped to 0 during cerebral hypoperfusion.

10.  For donation after cardiac death (DCD) kidneys, prolonged severe hypotension was a good predictor of subsequent organ function.  Donor age also correlated with worse outcome.

11.  Dogs anesthetized and hypovolemic shock induced for 2 hours.  NMR used to monitor phosphate metabolism.  Upon fluid resuscitation, phosphate pools quickly returned to near baseline values, but intracellular acidosis persisted.

12.  Hemorrhagic shock combined with increased ICP is particularly damaging.  Increased ICP leads to cerebral ischemia which causes release of thromboxane A2 (TxA2), a potent vasoconstrictor and hypertenstive agent.  The increase in TxA2 persists for at least two hours after reperfusion and results in further cerebral hypoperfusion.  Pretreatment with COX inhibitor ibuprofen decreases TxA2 levels and improves total cerebral blood flow after global cerebral ischemia.

13.  Brain is vulnerable during hypotension and shock, especially long-lasting shock.  Patchy areas of ischemia developed through sludge formation and persisted even after hyperperfusion, indicating the role of local factors.  Phenoxybenzamine pretreatment significantly reduced rCBF changes during shock.

14.  DCD livers result in inferior graft survival compared to donation after brain death (DBD).  A DCD risk index was developed.  The lowest risk is with donor age <= 45 years,  warm ischemia time (DWIT) <= 15 minutes, and cold ischemia time (CIT) <= 10 hours.

15.  CNS activity was measured during hemorrhagic shock under light central anesthesia.  After reinfusion, if neurons failed to recover electrical activity, this was an early indication of eventual irreversibility.  There is a relationship between irreversibility and cumulative oxygen debt and excess lactate.

16.  Rats were subjected to hypoxia and hypotension followed by resuscitation.  Rather than the no reflow that the authors were expecting, they observed hyperemia in some areas for at least two hours.  They concluded that therapy aimed at increasing cerebral blood flow and oxygenation would be insufficient.

17.  Guidelines for controlled DCD are given.  DBD is superior.

18.  DCD score system is described.  Kidneys may benefit from therapeutic interventions before transplantation.

19.  Average values for basal respiratory functions in adolescents and adults.

20.  Severe hypotension causes brain damage.  Microvascular damage results in hemorrhage upon reinfusion.

21.  Prolonged agonal time did not influence kidney transplantation outcome when other variables were closely considered instead.  For example, elderly donors were not included.

22.  During hypovolemic shock, electrical activity and ICP was minimally altered.  The authors interpret this as a lessening of the role of the brain in the genesis and perpetuation of irreversible shock.

References

1: Cavus E, Meybohm P, Doerges V, Hoecker J, Betz M, Hanss R, Steinfath M, Bein B.  Effects of cerebral hypoperfusion on bispectral index: A randomized, controlled animal experiment during haemorrhagic shock.  Resuscitation.  2010;81:1183-1189.

2: Cavus E, Meybohm P, Doerges V, Hugo HH, Steinfath M, Nordstroem J, Scholz J, Bein B.  Cerebral effects of three resuscitation protocols in uncontrolled haemorrhagic shock: a randomized controlled experimental study.  Resuscitation.  2009;80:567-572.

3: Chen RY, Fan FC, Schuessler GB, Simchon S, Kim S, Chien S.  Regional cerebral blood flow and oxygen consumption of the canine brain during hemorrhagic hypotension.  Stroke.  1984;15:343-350.

4: Cowley RA, Attar S, LaBrosse E, McLaughlin J, Scanlan E, Wheeler S, Hanashiro P, Grumberg I, Vitek V, Mansberger A, Firminger H.  Some significant biochemical parameters found in 300 shock patients.  J Trauma.  1960;9:926-938.

5: Feldman RA, Yashon D, Locke GE, Hunt WE.  Cerebral tissue lactate in experimental oligemic shock.  J Neurosurg.  1971;34:774-778.

6: Fritschka E, Ferguson JL, Spitzer JJ.  Increased free fatty acid turnover in CSF during hypotension in dogs.  Am J Physiol.  1979;236(6):H802-H807.

7: Gregory PC, McGeorge AP, Fitch W, Graham DI, MacKensie ET, Harper AM.  Effects of hemorrhagic hypotension on the cerebral circulation.  II.  Electricocortical function.  Stroke.  1979;10:719-723.

8: Hamar J, Kovach AGB, Reivich M, Nyary I, Durity F.  Effect of phenoxybenzamine on cerebral blood flow and metabolism in the baboon during hemorrhagic shock.  Stroke.  1979;10:401-407.

9: Hemmerling TM, Olivier JF, Basile F, Le N, Prieto I.  Bispectral index as an indicator of cerebral hypoperfusion during off-pump coronary artery bypass grafting.  Anesth Analg.  2005;100:354-6.

10: Ho KJ, Owens CD, Johnson SR, Khwaja K, Curry MP, Pavlakis M, Mandelbrot D, Pomposelli JJ, Shah SA, Saidi RF, Ko DSC, Malek S, Belcher J, Hull D, Tullius SG, Freeman RB, Pomfret EA, Whiting JF, Hanto DW, Karp SJ.  Donor postextubation hypotension and age correlate with outcome after donation after cardiac death transplantation.  Transplantation.  2008;85:1588-1594.

11: Horton JW, McDonald G.  Heart and brain nucleotide pools during hemorrhage and resuscitation.  Am J Physiol.  1990;259:H1781-H1788.

12: Kong DL, Prough DS, Whitley JM, Taylor C, Vines S, Deal DD, DeWitt DS.  Hemorrhage and intracranial hypertension in combination incresae cerebral production of thromboxane A2.  Critical Care Medicine.  1991;19:532-538.

13: Kovach A, Sandor P.  Cerebral blood flow and brain function during hypotension and shock.  Ann Rev Physiol.  1976;38:571-596.

14: Lee KW, Simplins CE, Montgomery RA, Locke JE, Segev DL, Maley WR.  Factors affecting graft survival after liver transplantation from donation after cardiac death donors.  Transplantation.  2006;82:1683-1688.

15: Peterson CG, Haugen FP.  Hemorrhagic shock and the nervous system.  1. Spinal cord reflex activity and brain stem reticular formation.  Annals Surgery.  1965;485-496.

16: Proctor HJ, Wood JJ, Palladino W, Woodley C.  Effects of hypoxia and hypotension on oxygen delivery in the brain.  J Trauma.  1979;19:682-685.

17: Reich DJ, Mulligan DC, Abt PL, Pruett TL, Abecassis MMI, D’Alessandro A, Pomfret EA, Freeman RB, Markmann JF, Hanto DW, Matas AJ, Roberts JP, Merion RM, Klintmalm GBG.  A J Transplant. 2009;9:2004-2011.

18: Plata-Munoz JJ, Vazques-Montes M, Friend PJ, Fuggle SV.  The deceased donor score system in kidney transplants from deceased donors after cardiac death.  European Society Organ Transplant.  2010;23:131-139.

19: Shock NW, Soley MH.  Average values for basal respiratory functions in adolescents and adults.  J Nutrition.  1939;143-153.

20: Tamura H, Witoszka MM, Hopkins RW, Simeone FA.  The nervous system in experimental hemorrhagic shock: morphology of the brain.  J Trauma.  1972;12:869-875.

21: van Heurn LWE.  Prolonged agonal time–not a contraindication for transplantation.  Nat Rev Nephrol.  2011;7:432-433.

22: Yashon D, Locke GE, Bingham WG, Wiederholt WC, Hunt WE.  Cerebral function during profound oligemic hypotension in the dog.  J Neurosurg.  1971;34:494-499.

“Synaptic”

As you wrote in 1994, the three sources of damage to cryopatients are 1) the underlying disease process, 2) shock and global and trickle flow ischemia secondary to dying and cardiac arrest, and 3) cryoprotectant toxicity and cryoinjury from freezing. This, as far as I can tell, has not changed. So, a flaw in how cryonics is practiced would have to mean that providers are not minimizing the damage from these processes as well as they could be. #1 is out as that is not the primary mission of cryo providers, although I agree with the arguments on your blog that they could add some value here too. #3 is also basically out, because gains over M22 seem unlikely to come in the near future, at least outside of 21CM.

That leaves #2. A number of the papers you sent me study animal models of hemorrhagic shock, and the results are not pretty for preservation of cellular structure. For example, the amount of necrotic cells in Ozkan et al’s paper is pretty high–up to 50% necrotic in the temporal lobe, after just 3 hours. The natural question is: if a cell undergoes necrosis, has it irretrievably lost the information coded in its cellular state? The answer is unclear. On one hand, it may be possible to reverse engineer the process of cell degradation from the surviving clues and thus recover the position of crucial membrane receptors and/or neurites. On the other hand, if the degradation process is random enough, that may not be the case. Probably it depends on the specifics — “cell necrosis” is a broad class.

A number of the other papers look at the acceptability of donors who died of cardiac death. It seems that kidneys can last up to 4 hr’s of warm ischemia with similar function post-transplant, while lungs following can hardly withstand 15 mins of warm ischemia time and still offer good function post-transplant. Meanwhile, it is practically common knowledge that the organ which is least able to survive following ischemic time is the brain. Finally, there is regional susceptibility variation within the brain, and there are reasons to think that regions like CA1 that may be especially important for identity (i.e., memory) are especially vulnerable to ischemia.

To me, this emphasized how quick the interventions must be and how essential it is to maximize the time period during which oxygen perfusion in the brain is high. There’s no reason why neurons have to be able to withstand lack of oxygen for long before randomly decaying — evolution has little reason to select for it. It is a bias of operating on human timescales to think that not much can happen within five minutes, but molecular timescales unfold much faster.

You also sent a few papers that evaluated measures to query brain activity via EEG. You seem to have a particular interest in one EEG-derived algorithm called the Bispectral Index, which in a few fascinating cases actually went to zero in the absence of cerebral blood flow during surgery. These are interesting in part because they could potentially be used to monitor CBF in cryo patients.

Which brings me to the major problem that we see in many of the case reports you sent me. That is, we have good reason to believe that all of them had already experienced a very low brain oxygen perfusion prior to clinical death. The signs of this are many, and include the hyperventilation of A2435 and A2361, the terrible peripheral perfusion of A1556, the hypotension and fluid loss of A1614, ACS9577′s poor perfusion and very low coma scale score, and the long periods of apnea and low blood pressure of A2420. One of the papers that you sent me called the period after removal from life support and cardiac death the “agonal phase”, and this phrase has been aptly used in cryonics to describe the period during which a patient is known to be eminently terminal but has not yet reached cardiac death.

One key question is whether these patients are ever in fact technically brain dead, meaning no neural activity at all, as measured by EEG or CT. If they are, then it is possible that clinical death could be pronounced and preservation techniques could be started much sooner. When I first thought of this, I was hopeful that I had discovered your “problem.” But on further contemplation I’m not so sure, in part because it seems like people would have thought of this. So, I am going with the more obvious, and indeed in some senses more troubling, problem that many or most cryonics patients experience torrents of brain damage during their agonal period.

What to do about this?

1) Somehow establish, in the US, legal recognition of the rights of cryo patients to initiate procedures to preserve brain-encoded identity when the patient is diagnosed by independent physicians to be terminal, in a similar way that organ transplants are.

2) Use a workaround by going to a country like Switzerland that already allows assisted suicide in such cases, perform the cryopreservation there, and then ship the patients back on dry ice to the US.

3) #2, except establish a new storage facility in the foreign country.

4) Develop, drawing off of the “normal” biomedical literature, substantially improved methods for preserving brain oxygen perfusion in agonal cryonics patients, and implement these on a routine basis.

One of the interesting things about this problem is that it is not specific to cryopreservation but would also apply to plastination, and may even be more pronounced there. So this is one area where progress, if any is made on either front, would certainly be synergistic.

A meta thought of mine about this assignment is that I didn’t like the assumption that I would be able to diagnose problems and suggest solutions so quickly to a problem that many people have spent lots of time thinking about. I doubt that what I have written above is at all novel.

Still, I did find it to be a very worthwhile exercise to learn about some details of cryopreservation and its associated medical concepts, and for that, I thank you for offering it to me.

——————————————————————————————————

I want to extend a sincere thank you to all who participated in this exercise, and especially to Alexander McLin, Mark Plus, Gerald Monroe, Jordan Sparks, DMD, and “Synaptic” for publicly participating. It takes an enormous amount of courage to undertake such an exercise on the Internet, where it both is and will remain open to public scrutiny, more or less indefinitely. Congratulations gentlemen, you have my unreserved admiration for your courage and for your willingness to take a personal risk in pursuit of the truth. — MD

Footnote

Screening for the Risk of Deanimation

The term “screening” is used in medicine to describe routine examinations or diagnostic procedures of a defined group of individuals to identify diseases or risk factors for same at an early stage. Screening is usually categorized as a  “preventive medical examination” or a  “checkup,” and its aim is to increase the life expectancy of those examined  by reducing the incidence or severity of life threatening disease and enhancing the quality of life. The most accurate examination methods possible should be used to identify as many diseases as possible still in their non-symptomatic phase, so that early treatment or change in life style can be initiated.

It is critically important to understand that the purpose of a “deanimation screening scan” (DSS) is not primarily to interfere with the course of disease or to extend the duration of life during this life cycle. Rather, it is to predict or to warn of impending  deanimation with increased accuracy and precision. Any contemporary medical or health benefits are thus incidental. Indeed, it is precisely when DSSing is used to determine or influence current medical interventions that it becomes dangerous. Knowing when you are likely to deanimate with greater precision, for sole purpose of improving your cryopreservation, carries little if any risk of iatrogenesis beyond that which would be present if you found out you were dying at a later time, or didn’t find out and suddenly collapsed in cardiac arrest from a heart attack, or suffered a massive stroke. It is only when the course of treatment is altered by obtaining the data, or looking at it (see “The Black Box of the Baseline,” below) that DSSing becomes either a practical or an ethical conundrum.

The first problem we confront in a screening test for deanimation risk is that we are moving in completely uncharted waters. We have no benchmarks or baselines on which to structure our screening program, save for a modest number of pilot programs that have been undertaken to evaluate full body scanning as a primary tool for the detection of cancer and atherosclerosis in the general population, or in selected subpopulations. For now, these will have to serve as the basis for our protocols, as well as the important cautionary lessons learned from other screening programs.

For reasons of safety, (see Radiation & Risk, below) Magnetic Resonance Imaging (MRI) is preferred over Computerized Tomography (CT), because no ionizing radiation is employed in making the image. MRI has some important limitations at this time, most notably only a few centers have devices that image the coronary vessels with sufficient precision  to allow risk  assessment for coronary artery disease (CAD).  Similarly, screening for Alzheimer’s Disease (AD),(beta amyloid deposits) also requires CT-PET scanning and the associated exposure to ionizing radiation.  So, for the present, CT is the only way to screen for CAD and AD. For this reason, and for those who for economic reasons may need to use CT imaging, it is worthwhile to briefly discuss the much hyped “risks” of radiation from whole body CT scans and this is done in some detail below.

Figure 25: Typical finding in an elderly woman who under prophylactic full body MRI scanning during a clinical trial in Germany to determine if full body scanning would reduce morbidity and mortality from cardiovascular disease and cancer. (Gohde, et al.)

A specimen imaging protocol is presented as Appendix 1 and is taken from the study by Gohde, et al., “Prevention without radiation – a strategy for comprehensive early detection using magnetic resonance imaging,” which was itself a pilot study in the use of MRI as a screening tool for cancer and cardiovascular disease.

The Mechanics

Currently, there is only one way to get a  DSS and that is to do it yourself.  There are several reasons, which will be discussed directly, why that is not a good idea, or certainly not the ideal way  to pursue DSSing. There are a number of reasons for this, starting with the potential for harm. Primum non nocere is the first dictum in medicine: first do no harm. Information is the most powerful force in the universe and information concerning you own health and welfare is especially important. It is also information that you cannot be objective about. It just isn’t possible. It is for this reason that no good physician treats himself or his immediate family in life or death matters as the sole or usually even the primary caregiver. In fact, speaking from experience as a person knowledgeable in medicine, I have found that wise counsel and advice I can (and do) easily give to others  is strangely absent from my own ears when I am the patient.

This lack of objectivity is more than a nuisance, it can be truly dangerous; and here I will have recourse to an actual example. The first four people to undergo DSSing have done so over the past 11 months. These were all individuals who were over 60 and who had not had consistent (or recent) “physicals.” All were counseled about the dangers of VOMIT and about the negative psychological impact of potentially finding out “something was wrong.” All four individuals had significant anomalies on their scans – two of which were life threatening and these were (or are) being medically managed.

In the other two cases, the scans revealed anomalies that might merit further medical evaluation in testing, and in both cases, the decision was wisely made not to pursue those tests. Why? That’s a complicated question, and I’ll answer it by explaining the circumstances of one of these people:

Mr. Ling is an 82 year old man who is in excellent health. He is physically active, mentally sharp and still working part time in his profession of many years.  He underwent a DSS five months ago. The findings were, overall, very good. His coronary calcium score was roughly a third lower than expected for his age, he had no signs of neoplasms, or of peripheral or central atherosclerosis, and the only abnormal cardiovascular finding was evidence of mitral valve regurgitation, which was deemed not serious and not likely to progress rapidly. However, a number of nodules were found in his right lung, along with some enlarged lymph nodes. The radiologist who reviewed the scan suggested a possible biopsy, with or without “bronchoalveolar lavage” (BAL).

While Mr. Ling is in good health, he is an 82 year old man and BAL requires sedation with propofol or a similar drug, and carries with it the risk of significant complications.  As to a CT-guided needle biopsy of the lung masses or the lymph nodes, this is this discussion that took place between Mr. Ling and the radiologist who interpreted his scan: “OK, let’s consider what this could be? I’m not sick – never felt better, so it’s not TB or something infectious? And if it’s cancer, well, what kind of treatment options would I have at my age for lung cancer with lymph node involvement?”

Those were great questions, and as it turned out, the radiologist was only playing it safe – he doesn’t want to get sued if Mr. Ling finds out he has cancer and a lawyer says to  a jury, “The doctor who imaged him said, ‘You’re in you 80s, I see this kind of thing all the time. Don’t worry about it.”  The radiologist ended by noting, “Since you are planning on following up in a year with another scan, we’ll see if anything has changed then.” And Mr. Ling is fortunate to have sufficient financial means that if he wants to pop in for a scan two months later, he can do that, too.

The problem is, most people aren’t in Mr. Ling’s position, and many will be unable to reason their way past the information that they have “masses” or “lumps” in their lungs and “enlarged lymph nodes in their chests!” That kind of worry cannot only be expensive, it can be damaging to one’s health, and corrosive to one’s quality of life. The information from DSSing should be given in the proper context, in the proper way, by the proper people, with the proper knowledge.  Absent that, it can do real harm. And if the scan does reveal a grave or untreatable medical condition, then there is all the more reason for the person to have the necessary resources at hand to help him cope and plan.

Ideally, this program would be part of a comprehensive Member Survival Program (MSP) administered by the cryonics organization (CO) and there would be a staff person whose job it would be to maintain communications with members, encourage compliance with MSP protocols (including the preferred imaging protocol) and collect and manage the resulting data stream.

Under such a scheme, upon intake (approval of cryopreservation arrangements) all members would have (at their option) completed a comprehensive health history and demographic information questionnaire, most of which would be completed as part of their membership application. The data from this questionnaire, as well as any electronic medical records the member may choose to provide, would be entered into the CO’s comprehensive member data base. The availability of this data would then allow for downstream refinement of the “one size fits all” scan protocol being proposed here, by allowing for individual risk assessment for CVD and cancer. This would flag members at elevated risk of early onset of these diseases to consider commencing scanning surveillance at an earlier age.

The Schrödinger Scan: the Black Box of The Baseline

Unless otherwise indicated, the first (baseline) scan would be done at age 45 for men and age 50 for women. In order to completely avoid any deleterious negative psychological effects, as well any potentially harmful effects from VOMIT (as discussed above), the baseline scan remains blinded and unexamined for 1 year after it is made. This done by providing written instructions to the radiologist reviewing the scan to seal the report unless there are unequivocal findings of life threatening pathology.

At the end of the year long blind period, the scan is examined and any anomalies noted. If the member chooses, a repeat scan can be done to resolve any questions or concerns raised by the baseline imaging. For example, if what appears to be a suspicious mass or nodule was found, a rescan a year later will very likely disclose if it is a neoplasm e.g., it will have grown or spread). It may seem counter intuitive to not look at data which you have paid for, experienced inconvenience to get, and which “might” save your life, but that is the necessary price that must be paid for this intervention to be used safely.

The baseline scan must be regarded as the first part of something that will not “happen,” or be completed for another year – like a bulb that has been planted to bloom in the spring, or a bond that will not mature for another 12  months. The scan itself is only a part of the process: the necessary information to safely interpret it does not appear until the required interval of time has elapsed. After all, before this protocol was proposed, no one ever got scanned and they felt just fine about it (until they dropped over in cardiac arrest).  For those of a quantum bent, consider it an extended version of Schrödinger’s famous experiment, except instead of the cat in the box, it’s a CAT scan in the box.

Scan Intervals & Exceptions

If the baseline is “negative,” showing no evidence of evolving pathological processes that merit intervention or further monitoring, then it is being proposed that the next scan take place 5 years later. Similarly, with each subsequent negative “healthy” scan, the next scan would be 5 years hence until age 81, at which point scans would be done every 2 years until cryopreservation ensues.

Figure 26: Proposed algorithm for Deanimation Screening Scan intervals and actions.

These scan intervals are arbitrary and will no doubt need to be refined over time as experience is gained. Intuitively, it seems that there should be a relationship between scan intervals and increasing age, and it is possible to configure scan intervals based on things like increasing risk of SCA or terminal illness with age. However, until some real world experience is gained, a conservative approach which minimizes costs and maximizes the opportunity for benefit, seems best. There are lots of programmers, mathematicians and similarly qualified people in cryonics and if any are interested in working with me, I am interested in generating scan interval algorithms based on the rising risk of disease and death with age (if you are interested, contact me at m2darwin@aol.com)

Going it Alone?

If a decision is made to proceed with DSSing on an individual basis, there are a number of important things to keep in mind and to do:

* Do consider carefully the possible impact this decision will have on you and on your family. In fact, give some thought to discussing this with your spouse or significant other before moving ahead.

* Do select a good imaging center with competent and caring staff who can give you good counsel about the procedure and the results. Imaging centers that offer full body scans are often used to counseling patients: make sure the one you select is a good one. Talk with the staff about your concerns before you commit to being imaged.

* Do explain to the radiologist who will interpret your images that you are having a baseline scan done and you only want to know if there is unequivocal pathology present that requires immediate or urgent medical intervention. If you can’t get that assurance from him, ask for your results only in writing on the same disk on which your scan is written.

* Don’t look at your scan or the written report that accompanies it. If you have a reliable and willing CO, send a copy to them and ask them to send you the results a year from when they receive the media with the images and the report on it. Duplicate CDs are typically made and given upon request at no charge, or for a small fee at the time you are imaged, or when you come for your results. Bring your own media to save money!

* Do provide a copy of the disk with the scan on it to your medical surrogate and to anyone who is on you ICE (in case of emergency) contact list on your mobile phone. The reason for doing so is that, should you experience SCA during the blinded waiting period, the scan may still save you from autopsy if it documents the presence of CAD, or some other pathology that could have caused your sudden and unexpected deanimation.

* Don’t  rely on the DSS to keep you out of trouble, or to reassure that everything is OK, should you develop serious health concerns. Just because a scan shows no indication of pathology does not necessarily mean that there is none. If you have signs or symptoms that would have prompted medical attention absent scanning, act on them in the same way after scanning. Let your physician decide if the scan is significant in the context of any illness or concerns.

* Don’t forget that the scan intervals are 5 years and that is more than enough time for serious disease to develop. Indeed, the 5 year window is a long one, especially where cancer is concerned. A DSS is not a health promotion or a disease prevention program. It’s primary purpose is to let you know you are terminally ill, not to assist you in avoiding that eventuality.

* Do know that if you have atherosclerosis, “vasculopathy” and you want to monitor progression of the disease, your scan intervals will have to be much shorter than 5 years – probably 6 months to 1 year, depending upon the severity, your response to medical intervention, and so on.

Economies of Scale?

Medical imaging is a highly competitive, non-monolithic industry consisting of many operators, large and small, both independent and institutionally affiliated. Such market environments inevitably encourage the drive to survive, and thus typically offer the discriminating consumer the opportunity for real bargains. I made a number of calls to imaging centers around the US and discussed the possibility of group discounts and “scan plans” wherein members of an organization or group, even just a group of like minded individuals, could get deep discounts on scans. The majority of centers I spoke with were receptive to this idea, and several discussed specific numbers which were anywhere from 20% to 60% lower than their standard walk-in fee.

Thus, it should be possible for groups of cryonicists in a given geographical area to make arrangements with a local imaging center for scans. The same was also true when I inquired about group or institutional discounts for carotid and abdominal ultrasound screenings, with the difference being that in some cases, prices went from ~ $350 per screen to ~ $60 per screen, providing the group could be scheduled for the same time and place.

The Pre-Cryopreservation Baseline CT Scan

Figure 27: A hypothetical pre- and post-cryopreservation  CT cerebral angiogram. The post-perfusion image would be obtained by administering radiocontrast agent(s) into the perfusate immediately, or shortly before discontinuing cryoprotective perfusion, prior to deep cooling to storage temperature.

If it is at all possible, a final vital CT scan of the head (at least) should be done as close to the time of cryopreservation as possible. This scan should be done with contrast and with no concerns about clinical radiation dose limitations, since the member will be terminal. The objective of this scan is to document, in as much detail (highest resolution) possible, the morphology of the brain and its vasculature. The imaging technique used should be one that optimizes resolution of the cerebral angiogram. The reason for making these images is that they should allow for many important determinations about the quality of initial stabilization and cryoprotective perfusion and cryoprotectant distribution in the brain to be made, at leisure, during the period the patient is in storage.

If contrast agent(s) is injected into the perfusion circuit shortly, or immediately prior to the discontinuation of perfusion, it should be possible to obtain a post-vitrification angiogram, which in turn should allow for evaluation of cerebrovascular patency, as well as assist in determining the anatomical landmarks within the cryopreserved tissue. It should also be possible to add other kinds of tracers to the perfusate, which might allow for quantification of regional distribution of cryoprotectants, or of other molecular species of interest not only within the brain vasculature, but within the brain parenchyma, as well. Again, the presence of a baseline pre-cryopreservation scan will likely be of great importance in allowing accurate interpretation of post-cryopreservation images.

This scan must be a CT, as opposed to an MRI, since MRI scans are unobtainable in deep hypothermia, or in the solid state.

Radiation & Risk

When the mass media talk about the “risks” from radiation associated with CT scanning, the first question that should spring to mind is, “Risks to who?” Sensitivity to ionizing radiation varies based on the cell age and mitotic cycle, and what this means in practical terms is that the younger you are, the greater the risk radiation presents to you.  Children thus have a much higher relative risk when compared to adults due to their rapid cell division and cell differentiation rate.

Figure 28: The risk of developing cancer as a result of radiation exposure is strongly age dependent and decays dramatically as people age. By the time an individual is in his 60s, 70s or 80s, the risk of neoplastic disease from medical imaging becomes negligible. Adapted from ICRP Publication 60 (1990).
 

These data also demonstrate that you cannot simply use the average relative risk shown in Table 1 to estimate the increased incidence of cancer due to radiation exposure. In order to do this analysis correctly, you need take into consideration the age of all individuals in the irradiated group. For instance, a man of 80 has a life expectancy of about 8 years, versus 33 years for a man of 45. Thus the risk to individuals over the age of 70 is, for all practical purposes, essentially nil. Table 2 illustrates what the  American College of Radiology considers minimal to high radiation doses in “absolute” terms.

This is why there is an increase in the relative risk values for the “entire population”  if children are included in that evaluation. However, even a quick glance at Figure 28 (above), where the estimated lifetime risk that radiation will result in cancer (carcinogenesis) is presented relative to the person’s age, shows that children have a 10% – 15% lifetime risk from radiation exposure, while individuals over the age of 60 have minimal to no risk (due to the latency period for cancer and the person’s life expectancy).  The accepted latency period is, by the way ~ 10 years.

Table 1 shows the relative risk of developing cancer per sievert (Sv) unit of radiation exposure. Tables 3 and 4 provide some comparison benchmarks of radiation exposure both in relative terms (low, medium, high) and in terms of common, specific medical imaging procedures used in regional CT.

So, let’s put this information in the context of a cryonicist wishing to reduce his risk of unexpected deanimation. The protocol being proposed here assumes a baseline scan at age 45 for males (50 for females) which, if free of any indication of ongoing morbid processes, is to  be repeated in 5 years, at age 51. If than scan is negative, subsequent scans would be performed at intervals of 5 years (if negative) until age 81, at which time the scan interval would decrease to 2 years. If we assume a lifetime cancer risk of approximately 1 in 1000 and a total of 7 scans  until age 81, at which point any further risk from radiation exposure becomes irrelevant, we might expect to see an increase in the lifetime risk of cancer from approximate 33% to 34%.  Even if the number of scans were more than doubled to 20; one per two years during the interval between age 50 and age 80, the lifetime risk of cancer would increase at most to ~ 35%.[1] This of course, assumes that all DSSs are CT, as opposed to MRI.

These risk calculations are based on the linear no-threshold (LNT) model of radiation risk.  This model assumes that the carcinogenicity of radiation is proportional to dose, even down to the lowest levels.  No one really knows how carcinogenic low-dose radiation is, because the carcinogenicity of low doses is so small that it’s practically impossible to measure. The official position of the Health Physics Society is that quantitative estimates of risk for doses below 50 mSv per year (100 mSv lifetime) cannot be made.[2]

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As useful aside, if you are interested in the progress being made in medical imaging, I would highly recommend the blog Magnetic Resonance Imaging: To See and Be Amazed: http://limpeter-mriblog.blogspot.com/ The site contains many beautiful images and is a treasure trove of information on both the mainstream progress, and the esoterica of MRI

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End of Part 4

(ischemia) ~85% of the time!

By Mike Darwin

Removing a Central Objection to Cryonics

In case you missed it, what I just said in that slim paragraph at the end of the preceding part of this article has profound implication because it has the potential to remove what is unarguably one of  the largest and the most rational objections that there are to cryonics. That objection is that roughly two-thirds of those who have made cryonics arrangements will not be cryopreserved under good conditions, and that half of all those signed up will be cryopreserved under very adverse conditions, such as autopsy or long (greater than 12 hours) post cardiac arrest delay. The recent advances in non-invasive medical imaging I’m about to discuss here offer the opportunity to we cryonicists to make many, if not most such losses all but unnecessary.

Figure 17: False color CT 3-D reconstruction of a patient’s intracranial arterial vascular tree. The orange-red, cheery shaped anomaly behind the right eye is a large aneurysm. The brain and other intracranial soft tissues have been digitally subtracted to facilitate a complete and unobstructed view of the patient’s arterial vasculature.

The image that you see in Figure 17 is now a perfectly pedestrian medical image that can be obtained from a garden variety CT scanner available at most diagnostic imaging centers in mid-sized cities anywhere in the world. This particular image has the brain, the soft tissue and everything digitally subtracted from it but the patient’s arterial tree and skull. The cherry shaped protrusion on the right is an aneurysm which, if were to rupture, could cost the patient his life or leave him profoundly disabled.

Figure 18: Many brain aneurysms can be treated non-surgically by passing a very thin platinum wire within the aneurysm where the wire coils up to form a yarn-like ball inside the weakened, ballooned-out area of the vessel wall. A clot subsequently forms around the coil and the vessel eventually closes off the opening to what was once the aneurysm.

Fortunately, there is a procedure  called “coiling” (Figure 18) which allows most such aneurysms to be successfully treated. Sadly, very people with brain aneurysms know that they have one until it ruptures – by which time it is almost always too late treat it effectively.

Scan Your Troubles Away?

The question logically arises, “Why not look inside everyone’s head if we have the technology to do so? Wouldn’t that allow us to identify not only the people who have aneurysms they don’t know about, but also everyone who has a tumor, or a narrowed coronary or carotid artery, or a gallstone, or anything else wrong with them that they don’t know about? In fact, why not scan their whole bodies and see if anything is amiss? Wouldn’t that allow us to nip most slowly progressing degenerative diseases in the bud?”

The answer to that question is a qualified “Yes and no.” The first and most important qualification to consider is the very substantial difference between them and us. They are going to die and, hopefully, we are not. Once you are content to die, it doesn’t really make a great difference exactly how it happens and it certainly doesn’t make any difference what happens to you afterwards. They will pay exactly nothing to avoid laying around dead for x-hours, or to avoid being autopsied. We, on the other hand, will pay something. That is a huge divide, because, as it turns out, the first and greatest barrier to such universal screening using CT and/or MRI is its adverse cost to benefit ratio.

Figure 19:  The rapid advance of computing and the high demand for ever more sophisticated medical images has driven the cost of 3-D CT and MRI scanning down to ~ $200 for a head scan $800 for a whole body scan.  http://www.superiorbodyscan.com/?gclid=CP_d5Neyiq8CFWwGRQodsHQX-w

While there are many CT and MRI machines, they are kept adequately busy, or perhaps just a little less busy than some of their owners would like, imaging sick and the worried well or hypochondriacal people. If the entire population, or even some modest fraction of it were to suddenly present for imaging, the system would crash. CT and MRI machines are very expensive and while the cost of scans has dropped dramatically, they are still not free. On the macro-level, governments, insurance companies and economists are constantly struggling to determine which therapeutic and diagnostic interventions offer the best return for the money invested in them.

The Problems of Bite Back and VOMIT

Surprisingly, information obtained from diagnostic tests can sometimes not only fail to yield any benefit, in which the case the money spent on the test is wasted, they can also cause harm. A recent example of this, much in the news, is the Prostate Specific Antigen (PSA) test used as a screening tool for prostate cancer (Figure 20). (http://www.pbs.org/newshour/rundown/2011/10/psa-testing-controversy-reignites-over-screening-debate.html) The problem with the PSA test as a screening tool is that to be effective in that capacity it requires a fairly long baseline, a good deal of contextual information (the patient’s race, family history, medications, and so on) and it requires good clinical judgment as well as a ‘patient’ patient.

Figure 20: It was anticipated that the PSA test, used as a screening tool for prostate cancer, would significantly reduce both the morbidity and mortality from the disease. It has so far failed to do so.

A single high PSA reading, or even several, may mean nothing. Most often it is the trend, rather than the absolute number; this is particularly true for black men.  In short, it’s a test that takes a lot of time and thought to interpret and use well and as such is probably not well suited to mass screening where a “yes” or “no” answer is sought before proceeding to costly, invasive and possibly injurious further evaluation.  Yet another problem is that even when prostate cancer is found and treated, it turns out that very few lives are saved because most of those cancers are slow growing and in men who will die of something else before the cancer kills them. Thus, the cost to benefit ratio of the PSA is being questioned, not the least of which because it causes many men to suffer and even die from treatments from which they did not benefit!

This is very much where medicine is today with respect to the “medical imaging singularity.” While it is possible to “look inside” just about everybody, the cost to benefit ratio for the health care system and for the “man on the street” would not justify it. In fact, it would be a medical catastrophe.

To understand why this is so it is necessary to understand three things. The first and most important of these is something called VOMIT, which is a very serious form of bite back associated with our new found ability to see inside patients with increasing exactitude. VOMIT stands for Victim of Medical Imaging Technology and refers to patients who suffer unnecessary interventions for abnormalities observed by imaging or other investigational technology, but which were not found during surgery or subsequent invasive diagnostic interventions. (Hayward, 2003) Here, I will go further and extend the definition of VOMIT to include any diagnostic finding which result in a diagnostic or therapeutic intervention which is not cost effective or causes harm to the patient. That is a very important caveat and tall order to fill, as we shall soon see.

The second is the relatively straightforward one of the ratio of the dollar benefit of resources expended to dollar benefit returned in years of productive life saved as a result of the intervention. Even in cases where early diagnosis saves lives, such as in breast cancer screening, the economic returns are equivocal. It is also often the case that “early” diagnosis with existing imaging technology is still not early enough to cure the disease. As a result, the patient suffers a longer, more miserable course of treatment and the healthcare system is subjected to greater expense with no return.

The third is the problem of information overload and it is somewhat related to VOMIT. The truism that a picture is worth a thousand words is probably a vast understatement. A single 3-D medical image contains a vast wealth of information – information which has heretofore been unavailable to both the clinician and his patient.  This might seem like a good thing, and in the long run it will be, but for now, and for a long while to come the details of the landscapes being revealed will, to a great extent, be terra incognito.

The Danger of TMI

When advances in microelectronics allowed for 24-hour ECG monitoring in the 1970s,  it became possible for clinicians for the first time to see the beat by beat electrical activity of their patients’ hearts for up to a day at a time, or longer. Prior to that, they were limited by the enormous quantities of paper tracings that would be required and the need to confine the patient to the clinic or laboratory. Now, with the advent of the compact and mobile “Holter monitor,” it was possible to capture the patient’s ECG data continuously under ambulatory, real-world conditions (Figure 21). Physicians were awash in a veritable sea-tide of data!

Figure 21: The Model 445 Mini-Holter Recorder which was released in 1976 allowed clinicians for the first time to “see” their patients’ ECGs under real-world conditions and for prolonged periods of time.

The problem was , they assumed, quite understandably, that they knew what it all meant. After all, doctors had been looking at patients’ ECGs for decades in their offices, in hospitals, at bedsides in homes and in physiology laboratories. They knew how to read  an ECG! So, when they discovered that some of their patients had periodic bouts or “runs” of very worrisome arrhythmias, they did the prudent and rational thing – they treated them for these arrhythmias with medications. Unfortunately, the result was the opposite of that expected; a significant increase in morbidity and mortality in these patients, because it turns out that in a subpopulation of healthy people, those arrhythmias were benign and not indicative of any health problem.  Thus, misinterpretation of the “same” information they were confident in dealing with in small chunks, presented in bulk and in a different context, was one of the unforeseen and arguably unforeseeable bite back consequences of Holter monitoring technology. (Harrison, 1978)

The Last Heart Attack?

If you assemble and then read over the Alcor case summaries of the last 40 years it is impossible not to be shocked by the seemingly high incidence of sudden and unexpected cardiac arrests. Because my data set is incomplete for Alcor, I can’t be definitive, but the number seems to be somewhat higher than for the same subpopulation of people from the general population (white, middle class, etc). Until, that is, you consider that most cryonicists are male. So, as you read accounts of cryonicists in their 40s and 50s arresting while scuba diving, while taking a nap or watching television, in part what you are seeing is selection bias at work. The point is, no one ever died of “sudden heart disease” a “sudden aneurysm” or, for that matter “a sudden cancer.” These are degenerative disease that takes years to decades to develop. While still difficult to detect in their nascent stages, their terminal lesions are usually very visible many months and sometimes for even for many years before they end lives.

Figure 22: Coronary artery calcium scoring using computed tomography and carotid intima media thickness and plaque using B-mode ultrasonography offer the prospect of detecting almost all coronary artery disease before it reaches the stage where it can cause a heart attack or sudden cardiac arrest.

There has been a great deal of media attention lately to an initiative called SHAPE; The Society for Heart Attack Prevention and Eradication,  which aims to all but eliminate heart attacks by combining CT of the heart to obtain a “myocardial calcium score” (a powerful risk predictor of heart attack)(Figure 22) and carotid intima media thickness and plaque using B-mode ultrasonography as part of a three step program to eliminate heart disease. The next two steps in SHAPE’s plan are a “polypill” combination of blood pressure and anti-atherosclerosis drugs and finally, perhaps, a vaccine. A similar “Last Heart Attack in America” initiative focused on coronary scanning along with dietary interventions to reverse atherosclerosis has been the focus of a feature length documentary on CNN in which former US President Bill Clinton is prominently  featured as a spokesman and advocate. The common ground of these two initiatives is that almost no one dies of a heart attack without there being  glaring evidence present in their hearts years before the infarct occurs. It is only necessary to look for it!

There can be no question that as imaging technology evolves, and as medical acumen catches up with what is available, that such imaging will become a routine part of any checkup  for patients whose age and risk profile merit it (and eventually, if they live long enough, that means most patients). As it stands right now, if you are a middle aged man or woman with a significant risk profile for heart disease, and you have a heart attack, it’s my personal opinion you have ample grounds to sue your physician for negligence.  Right now, that’s just my opinion, so it doesn’t count for anything, but the point is that sooner or later this, or a better coronary imaging modality is going to become the standard of care and heart attacks will become a rare event – a thing of the past – a relic from a time when doctors couldn’t see inside of you.

Ultrasound Investigations

There are cheaper, simpler and completely risk free ways (in terms of radiation) to  find out whether you have atherosclerosis or not.  The most predictive of these for money is the carotid ultrasound (CUS) test.

Figure 23: The carotid ultrasound scan is  a simple, non-invasive diagnostic investigation that employs sound waves to create an image of the two large blood vessels in the neck that supply most of the blood to the brain. If there is a buildup of plaque or a thickening of the limning of these two arteries the person is at increased risk of stroke and there is a high probability that there is also systemic atherosclerosis present. If there is evidence of severe narrowing of one or both of the vessels, then it becomes urgent that medication and possibly surgery be used to correct the condition in order to avoid the likelihood of a crippling or lethal stroke.

This simple, non-invasive test takes just a few minutes and uses ultrasound waves to image the carotid arteries and the blood flowing through them (Figure 23). If there is thickening of the arterial wall, or plaque present, then it is a virtual certainty that the person has systemic atherosclerosis and warrants a more extensive workup. This test is often also “packaged”  with a quick “look-see” at the abdominal aorta also using ultrasound, to rule out the possibility of an abdominal aortic aneurysm – something that is more common in smokers once they reach middle age, and beyond.

If you shop around diligently, the cost a CUS can be as little as your transportation costs to the health fare or community center where it is being offered, often as a “loss leader” by health care providers or medical imaging companies seeking more remunerative business opportunities (if they find something amiss during the CUS).  The cost of such an evaluation can range from as little as $60, to as much as $380.

A CUS is ideal for people on a budget and for those under age 45 with no history of heart disease, cancer or other pathology or risk factors that might put them at increased risk of sudden cardiac arrest.

Why Full Body Scans?

Figure 24: The full body CT or MRI scan is often offered as “add-on” to the complete or the “executive’s” physical. Many imaging centers offer these scans without the need of the patient’s person physician prescribing the scan using their in-house radiologists to write the order for the test. http://www.prevenium.com/contact.asp

 Put simply, there is no substitute for seeing, or to put a new twist on an old adage: a picture is worth a thousand medical tests. While the origins of all of the degenerative diseases that kill us are at the molecular level, mostly we die as a consequence of the macro-level changes they inflict on our bodies, even if the coup de gras is rooted in the action of things like adhesion molecules and inflammatory pathways; as is the case with most heart attacks. It is the large, easily “seen” bulges of aneurysms, masses of plaque or tumor that kill, and these almost always take years to develop. What this means practically is that, with a few exceptions, aside from suicide, homicide and accident, virtually no one has to die – or to deanimate without plenty of advance warming. The implications for cryonics are as obvious as they are profound.

End of Part 3

(ischemia) better than ~85% of the time!

By Mike Darwin

Ischemia: The Problem of “Long Down Time”

 Almost every cryonicist I’ve ever spoken with envisions his cryopreservation will occur under ideal circumstances. He will be diagnosed with  some vague and ill defined terminal illness, bravely decide to end futile treatment and then enter hospice with a team of skilled and caring cryonics personnel at his bedside. He will nap, read, watch TV, and then, near the end, nod off surrounded by loved ones as the cryonics personnel hover nearby. This may not be the most attractive picture in any absolute sense, but it is certainly as reassuring a one as it is possible to find in contemporary cryonics. And while many, or even most cryonicists may find this scenario credible, much of the rest world doesn’t.

 Figure 10:  Approximate U.S. distribution of predictable deaths by cause based on 2004 data. Note that ~57% of all deaths occur sufficiently suddenly, or under circumstances such as accidents, which preclude standby or other cryonics stabilization measures. Chart derived from data: [National Vital Statistics Report, Volume 53, Number 5 (October 2004)]. This data may be compared to the data in Figure 10 to see how closely the US national incidence of sudden and unpredictable death map that of Alcor’s experience (Figure 11).

One likely reason for the scarcity of biomedical people involved in cryonics is that their actual, day-to-day experience is at sharp odds with the scenario I’ve just laid out above.  In countless hours of both focused and casual conversations with such individuals, what emerges is a sense of incredulity about the reversibility of the damage these professional and technical people witness as a part of their duties caring for the very old, and the critically ill dying; not to mention that large fraction of people who die suddenly and without warning, end up as DOAs in the emergency department or coroner’s cases. Regardless of whether their opinions prove the valid ones, we are clearly failing to communicate to them and to the community at large, an experience of cryonics which is not so biomedically adverse.

To do that, it is first necessary to move beyond  anyone’s scenarios or suppositions and evaluate the reality of what is actually happening to the patients we cryopreserve. That turns out to be a hard thing to determine with any degree of precision, because none of the cryonics organizations maintain any kind of statistical database on their members’ cryopreservations. How many cryopatients have dementia? How many were autopsied? What is the mean ischemic time from cardiac arrest to the start of cardiopulmonary support (CPS)? How many patients have ischemic times of 2-5 minutes, 5-10 minutes, 15-30 minutes, 12 hours, 14 hours, 5 days? What is the mean age at cryopreservation? [Absence of data on this last question I find particularly amusing in a group of people supposedly preoccupied with longevity and "life extension": how long are they living, on average?]  There is currently no way to tell.

There is not even any way to determine the age, gender, or any of dozens of other potentially critically important demographic details that are, or could be vital in assuring quality cryopreservations, reducing ischemic times, or reducing known iatrogenenic events. A concern of mine for onto three decades now is that we have no way to spot adverse epidemiological events that might be associated with our unique dietary supplement or other lifestyle practices. Perhaps most incredibly, there are no written criteria, however arbitrary, to assign any degree of quality, or lack thereof, to the cryopreservation a given patient has received (let alone that a given Cryonics Organization (CO) provides, on average). This had lead to what has become known as “the last one is always the best one” to date rating system, wherein each case that is not either an existential or an iatrogenic disaster, is pronounced by the staff who carried it out as, “the best case we’ve done so far!”

We cryonicists may be in some kind of willful, data free fog about what our situation is, however, it’s a safe bet to assume that most of the rest of the world, based on their own professional and personal experiences, are not so ignorant. The first step towards a solution is to understand the scope and severity of the problem by getting reliable numbers. While that is not easy to do, the Alcor Life Extension Foundation does maintain a crude, if incomplete accounting of all the patients they have placed into cryopreservation: http://www.alcor.org/cases.html. A cursory analysis of this yields the following breakdown. Even basic data such as cause and mode of death are missing from ~20 of the cases listed there – these have necessarily been excluded from the analysis below.

Figure 11: A major hurdle to evaluating quality in cryonics operations is the lack of any outcomes (e.g., reanimation followed by evaluation) or of any surrogate markers or scoring systems to serve as evaluation tools to determine not only the quality of cryopreservation care being given, but also the objective neurocognitive status of the patients when they enter cryopreservation. For the purposes of this analysis very crude criteria were used to assess the quality of the patient as a finished product at the end of cryopreservation. These were normothermic ischemic time between cardiac arrest and the start of CPS, catastrophic peri-arrest brain injury such as an intracranial bleed followed by prolonged cerebral no-flow before pronouncement of medico-legal death, very long warm ischemic times (> or = to 12 hours) and autopsy.

Using the criterion of “minimal ischemia” (≤15 minutes)[1], 48% of Alcor’s patients are cryopreserved under these conditions (Figure 10).  Thirty-nine percent of their patients suffer long ischemic periods of 6-12 hours or more (mostly as a result of SCA and UDA); and 13% suffer very long periods of ischemia (> or = to 24 hours) which are not currently preventable, or which conclude in autopsy prior to cryopreservation.  Put more cogently, you have less than a 50% chance of being cryopreserved (with Alcor) under conditions of minimal ischemia. While this number is discouraging, it is spectacular when compared to the Cryonics Institute, where it is somewhere in the low single digits.

Figure 12: The graph above is the same as in Figure 11, with the difference being that the losses have been expanded to include those that would be expected if the population wide incidence of end-stage, GDS-7 dementias were imposed on all the groups. The result is that percentage of patients who might reasonably be expected to have both minimal ischemia and no pre-cryopreservation GDS-7 dementias drops to just 26%.

But once again, these numbers are misleading if the criterion is cryopreservation under minimal ischemia conditions, because they do not take into account the number of patients who enter cryopreservation with dementia, or severe brain injury due to stroke, other neurovascular disease, or massive head trauma. If only dementia, at the current incidence for the general population is factored into the analysis, then the picture becomes considerably more bleak, as can be seen in Figure 10, with only 26% of  Alcor cryonics patients being preserved with relatively intact brains under reasonably good conditions.[2]

Impact of the BDDs on the Likely Survival of Personhood

Figure 13: The effect of advanced Alzheimer’s Disease on the macroscopic appearance of the brain is evident when coronally sectioned brains from an AD (R) patient and a healthy person in their mid-20s (L) are compared side by side.

Deaths from AD are typically deaths from end-stage AD, which usually implies severe global destruction of both cerebral hemispheres (Figures 13 & 14) on both a macro and microscopic level. Death due to AD is a prolonged process (~8 years from diagnosis to death), and the neurological deterioration that occurs as the disease progresses is often scored using the global deterioration scale (GDS) of primary degenerative dementias, which ranges from 1 (least) to 7 (worst) in severity. GDS scores in excess of 5 are associated with major loss of macro- and micro-scale brain structure and will be assumed here to represent serious compromises to, or the destruction of personhood.

Figure 14: The histological appearance of the brain in AD is shown in panels b and c above. In many areas of the brain there is virtually complete loss of the neuropil; the synaptic weave that interconnects neurons which can be seen in its normal state in c, the panel at the far left. The majority of the neurons and many of their supporting glial cells have died and been scavenged by macrophages and histiocyytes.  There are abundant deposits of proteinaceous plaque containing the  neurotoxin protein beta amyloid neurofibrillary tangles which are the remnants of neuronal long processes such as axons and dendrites. The extent and uniformity of the changes seen above varies from patient to patient during the course of the disease, but becomes increasingly uniform throughout both hemispheres of the cortex the longer the patient survives with a GDS score of 7 (end stage dementia).

A Deanimation Warning Device?

Figure 15: The medical imager as a deanimation prediction device?

 In his 1939 science fiction story Life-Line,” Robert Heinlein envisions a device that can predict, with considerable precision, when a person is going to die. While none of us cryonicists wants to die, most of us could certainly profit from knowing when we are going to deanimate. Better still would be also finding out how to postpone our cold dip in liquid nitrogen for a while, if it was possible to do so.

Many cryonicists will be familiar with this graph of Ray Kurzweil’s showing the impending arrival of the singularity (Figure 16).

Figure 16: Ray Kurzweil’s graph showing the exponential increase in neuro-image reconstruction which has occurred largely as a function of the exponential growth in computing capacity since 1970.

Well, if you are a cryonicist, I’m here to tell you that insofar as non/minimally-invasive medical imaging is concerned, the singularity is here.

From the earliest days of medicine physicians have desired one thing almost above all others and that is to possess the power to peer into their patients bodies and observe the goings on there. Since the discovery of x-rays by Wilhelm Conrad Röntgen in 1895 (Crane, 1964) there has been steady progress towards the satisfaction of that desire. The development of contrast media, endoscopy, computerized axial tomography (CAT or CT) scanning and magnetic resonance imaging (MRI) scanning have allowed increasingly exact and impressive images of the interior of the living body to be made.

However, a number of serious limitations have, and to a great extent still do prevent the full realization of the physician’s idealized desire to see inside his patients at will. Those barriers are field, dimensionality and point of view, as well as resolution, color, contrast and the dollar cost of the imaging.

In the case of CT and MRI those barriers have been breached to such a degree that it is now possible for cryonicists to be able to determine with a very high degree of accuracy and precision both of what and when they are going to experience medico-legal death. A corollary of this is that in many cases it will be possible for them to avoid what would have otherwise been an unavoidable very long period of ischemia and quite likely a medico-legal autopsy  as well.

End of Part 2

(ischemia) ~85% of the time!

By Mike Darwin

Foreplay

It has taken me roughly 30 years to learn that having the technological capability to achieve some marvelous end is only a small part of the battle to actually achieving it.  This is profoundly true in the world of biology and medicine because, unlike as was the case with “free speech” and “private life,” there was no Martin Luther and no Thomas Paine to definitively divorce these areas of human endeavor from the grasp of the religious moralists, the secular ethicists, and the social busybodies of the earth. The life sciences have yet to have their Martin Luther’s 95 theses nailed to the doors of the places in which this culture’s moral tyrants currently reside. The separation of Church from private life which began with Luther, and of private life from state, which began with the Magna Carta and the US Declaration of Independence, could take us only so far.

Now, we are in an interesting place and time, because never before have potentially lifesaving technologies been being generated at such a phenomenal rate. And yet, they remain outside our grasp as surely and solidly as if there were an impenetrable Prespex wall between them and us. We can look, but we can’t touch.

Beyond our physical inability – or seeming physical inability – to access those lifesaving capabilities, we also pay a heavy price in a different way. Our vision and perspective becomes warped. We literally become unable to see how we might help ourselves, because we have been conditioned to be dis-empowered. We lose the ability to think outside the box and we begin endlessly replaying the failed or marginal strategies that the existing system does allow us to pursue.

However, a close look at our predicament will reveal that that Perspex wall works mostly for the masses – for them – and not for us. If we are careful and clever, we can reach through it and extract much of the technological benefit sitting there. We can do this, but they can’t. Once we understand that, it has the potential to change our perspective on everything in terms of our chances for survival, and for our chances of living productively and in comfort, while much of the rest of world may well pursue a very different path.

That’s what this article, and the ones that follow it, are about. This article is preparatory, it’s a kind of foreplay to prepare you for the powerful penetration of the ideas that are to follow.

Of  Singularities & Hams

Figure 1: Jamón ibérico de bellota is a gourmet ham made from black Iberian pigs fed only acorns during the months prior to their slaughter.

 The first few times it happened, I hardly noticed, and I can’t remember the specifics. But when it really began to annoy me I can  remember, quite clearly, perhaps because I was already in a foul mood and the surroundings were extraordinary. We had been taken out earlier in the day to see the pigs from which the jamón ibérico de bellota is made. The vile, dusty, slobbering and altogether horrid beasts are fed nothing but acorns so that their flesh is rendered especially succulent and flavorful after elaborate smoking and aging. They were moving about with indifferent belligerence, unaware that their kin were to  be on the supper menu late that afternoon. The visit to their quarters made me thankful I did not eat land vertebrates and reminded me uncomfortably of some of my compadres at the Hacienda; the several “Mr. Bigs” who had gathered to discuss the creation of a new cryonics enterprise.

As we sat down to dinner in the courtyard of the Hacienda that evening, I was seated at a table with several middle aged cryonicists and two older ones, (sadly, including myself). It wasn’t long before I was bombarded with the question I would soon find irritating, and eventually come to loathe: “Have you had genomics testing done?”

Figure 2: The courtyard of the Hacienda where my dinner companions assailed me over my lack of diligence in having my genotype analyzed to determine my disease risks.

“And why would I have that done, I asked?” My questioner, an enthusiastic thirty-something, leaned forward a bit and explained to me how rapidly the cost of sequencing DNA base pairs was dropping, and that it was now possible to tell all kinds of things about an individual’s risk for diseases by genotypic analysis.

“It costs only  $200 US; I just had mine done.”

Others began to chime in. Since it was an international crowd, the stories were fascinating and I was content to listen. Some had discovered they had Neanderthal lineage, others had discovered less exotic, but no less unexpected genetic heritage. Finally, the conversation returned to me, the apparent elder statesman and, presumably, the example setting cryonicist at the table: why hadn’t I had my genotype evaluated, and much more importantly, why didn’t I have any plans to do so?

“Look, ” I said, “I think genomics  technology is going to be incredibly valuable. I think its most immediate value is going to be in pharmacogenomics – in determining which drugs work for which individual people and which drugs don’t work, or are actually dangerous for given individuals. A bit later, this technology will likely have real prognostic value. But not now, and not for me. I’m in my early-50s. My relatives are already sick, dying or dead of illnesses that are genetically mediated. I know what my genetic risks are. In fact, from my family history alone, I’ve known what those risks are for roughly 20 years now. Both my parents are now in their 80s, and I have a very good idea of what they are going to die of. And if they don’t die of those things, well, it will be from an accident, an infection or something not likely to be readable in the tea leaves of my genome.

 Figure 3: The Hacienda on the arid Spanish countryside outside Madrid where we took our repast and discussed singularities, past, present and future.

Interestingly, my parents have had every single disease that has also killed their parents, their aunts and their uncles: cancer, hypertension, atherosclerosis, alcoholism, type II diabetes, and Alzheimer’s Disease (AD). I’m pretty sure that AD is going to claim my mother’s life, and I’d say it is probably down to atherosclerosis, and possibly cancer or emphysema, in the case of my father. With the help of modern medicine, my folks have so far dodged all of the other genetically mediated bullets that have been shot at them. So, I know my genetic risks  (and to those I’d add the risk of some peculiar autoimmune diseases in late life are present in my maternal bloodline).

But by far my biggest risks, which would not yet (to my knowledge) show up on any genotypic test are Bipolar-2 Disorder and homosexuality, both of which have a devastating impact on longevity, dramatically increasing the risk of a broad range of pathologies, including cardiovascular disease, cancer, dementia, substance abuse, other mental illness, and all cause mortality. My point is that in most cases where genes influence destiny, you’re best clue is the evolved or evolving fate of your kin – unless you are an anonymous orphan, that is.”

Still, they wouldn’t give up. The implication was that I must have genomic testing. And, truth to tell, I had, and have, no objection to it. It’s not like I am opposed on religious grounds, as if it were fortune telling. “In fact, I think it’s a nifty conversation piece and personally interesting in the bargain. It’s just that I’d have a lot higher priority uses for my $200 in terms of the dramatic medical advantages it could buy me as a cryonicist, if I had $200 to spend on such things! It would make a wonderful Newton Day gift, the kind of thing you’d like, but would never buy for yourself.”

Now that, that statement really set them off! I had thrown gasoline on a fire. Didn’t I know that the exponential decrease in the cost of DNA sequencing constituted a Singularity in biomedicine, one that was, even as were sitting there that very moment, revolutionizing medicine? “Sure.” I said, “But  there are singularities happening all the time. The thing is, most singularities in medicine unfold over a period of decades, and very few individual patients gain benefit from them on the basis of special, unique, or insider knowledge.”

But, I had lost them. They were having none of it, and I wouldn’t be the least bit surprised if I’ve lost you as well. I was irritated and frustrated and I had already lost my temper badly earlier that day. So, I decided to bite my tongue and proceed in relative silence with the rest of the meal. But what I really wanted to say to those gentleman was that, “you wouldn’t know what to do if a medical singularity were to come right up here and bite you in the ass, because it already has!”

One of the (many) reasons the meeting had crumbled was the intransigence of one of the Mr. Bigs, who wanted cryonics with the stipulation that there be essentially no ischemic time. He had his approach to solving the problem which was, well, this meeting was some years ago, and I wonder if Mr. Big is still alive?

It was a strange situation. Mr. Big was clearly not a well man and he knew this to be the case. What I suggested was straightforward, involved nothing either exotic nor illegal and was something that I knew would work, based on the sorry experience of seeing it not work with men exactly like him. I tried to explain to Mr. Big that it was now possible to “simply” look inside of him, from top to bottom, and get a fairly accurate assessment of what his risks were for deanimating in the near future. Given his medical history, which he shared with me,  I also suggested that he have a condition treated which would, probably sooner rather than later, cost him his life, or leave him profoundly disabled. He was having none of that, either!

Instead, a few hours later, here we were seated together at dinner and Mr. Big was extolling the virtues of genomic testing as a way of avoiding premature cryopreservation-  to me.  A true, nearly unalloyed medical singularity had arrived for cryonicists, and for the previous two days they had snuffled and shuffled around each other with same indifferent belligerence of the hogs in the pen nearby who were awaiting their conversion to jamón and their journey away from the Hacienda in someone’s belly. It is at moments like this, which come with increasing frequency, that I sneak a quick look out of the corners of my eyes to see if I can catch a glimpse of some dimple or ripple in the fabric of reality that will clue me into the fact that my life has really been just a joke in very poor taste  – on me.

I’ve struggled mightily with how to effectively communicate the idea that for cryonicists, a singularity of truly incredible magnitude has arrived and that it is one which, in theory, should be available for use by us now. I’m reasonably sure I’ll fail in that task and that no matter how I might have framed the argument, or presented the evidence, the outcome will remain the same. And therein probably lies yet another powerful lesson about why Singularities, wherein everything is transformed in the blink of an eye, never really happen.

How ‘Fast’ are Most Medical Singularities?

Medicine, ironically  much more so than entertainment or warfare, is bound up with the sensitive issues of ethics and morality, which have historically complicated and often slowed the propagation of paradigm changing, or so called “singularity events” within its confines.  Vaccination, contraception, anesthesia, organ transplantation, mechanical life support, resuscitation medicine, in vitro fertilization and embryo and gamete cryopreservation have all been slowed or blocked altogether as a result of religious or ethical concerns. (1,2,3) Indeed, surf the net or turn on TV today and you will see hordes of angry people decrying vaccination, contraception, and arguing furiously over life support. Support for vaccination, ~212 years after Jenner, is even eroding in the nation that spawned it!

The idea that wound infections – sepsis – were caused by a contact-transmissible agent was definitely proved by 1848, in the form of the exhaustive statistical work documenting the effectiveness of antisepsis conducted by Semmelweis. By 1860, the theoretical grounding for the basis of that transmissible agent, germ theory, was in place. Scattered throughout Europe there were a few men who understood the new paradigm and could no doubt foresee many of its practical implications in medicine. These men must have been as frustrated as cryonicists in the middle of this last( 20th) century – men like Pasteur and Koch. If ever there was a singularity in medicine, this was it. And yet, what happened?

Figure 4: President (then General) Robert E. Lee of the Confederate States of America receiving his critical Magic Lantern briefing on the revolutionary, but heretofore unappreciated work of the Hungarian physician Dr. Ignaz Phillip Semmelweis, concerning the importance of antisepsis for the control of infections in battlefield and surgical wounds. The information proved of a vital strategic advantage in helping the Confederacy to successfully prosecute the war against Union forces. Lee is seen here in the sitting room of his home in Arlington, Virginia in this classic painting by John Elder.

Perhaps it might be more instructive if we ask ourselves what should have happened according to the Singulatarian, or even according to the “popular” model of how  powerful, beneficial ideas with virtually no downsides spread through the culture. For instance, one of the most popular “what if” questions in the realm of alternate history is, what if this or that had been different that would have altered the outcome of the United States Civil War?(4) Military historians all have their favorite “what ifs” in this regard, but mine, well mine wouldn’t be military at all, but would come down to a long, drawn out Magic Lantern (PowerPoint) presentation given to a very receptive General Robert E. Lee, on the eve of the Secession. The subject of that presentation would be the revolutionary findings of two maverick Europeans; Dr. Ignaz Philipp Semmelweis, and  Dr. Louis Pasteur, as they apply to battlefield medicine and the recovery and survival of injured troops in the conflict to come.  The Confederacy lost the war for many reasons, but in the end it came down to a lack of manpower and the disproportionately draining and depressing effect that combat related sepsis had on the South. [At least, that's my story and I'm sticking to it ;-).]

Lee would listen, his military surgeons would be briefed on the Confederacy’s “secret weapon” and the tide of history would be turned. Wild and playful imaginings? Yes, but they constitute a considerably more reasonable scenario for the rapid adoption of asepsis in the US (or even half of it!) than just about any other you are likely to come up with, because the reality of what happened is almost incomprehensibly tragic.

Figure 5: In his magnificent painting entitled The Gross Clinic, Thomas Eakins graphically captures the state of surgery in the US during the decades following the US Civil War. These grotesquely unsanitary conditions had by this time to a large extent become a thing of the past in surgical theaters through much of Europe.

Figure 6: Even 14 years later, when Eakins revisits the them of the operating theater in his painting The Agnew Clinic, full adoption of asespsis and antisepsis had not occurred in the US.

Semmelweis’ work had already been published and disseminated around Europe by 1848, and by 1861, the year the American Civil War was opening, Lister was reprising Semmelweis’ discovery of antisepsis in Scotland, not with chlorine, but with carbolic acid. The sad reality was that the Americans (North and South) were so pigheaded regarding germ theory and the value of asepsis and antisepsis to medicine, that it would not be until well into the 19th century before that particular singularity fully took hold of the United States.(5)

Indeed, Lister made an “evangelical” tour of US medical schools in 1876 to little avail.(6)  Whilst the Listerian revolution was well underway in Europe by then, the situation in the US was to remain, as it was so vividly portrayed by Thomas Eakins in his magnificent oil, The Gross Clinic, which was painted the year before Lister’s missionary visit to the germ loving heathens across the pond. Fourteen years later, when Eakins painted The Agnew Clinic, we can see the beginnings of asepsis just starting to take root in the form of basic cleanliness being imposed in theatre. Clearly, antisepsis/asepsis are an example of a technological singularity in medicine, albeit one that took onto a century to fully unfold!

The Problem of Bite Back

But beyond these arguably irrational roadblocks slowing the progress of technological singularities in medicine, there are two others: the very real problems of their rational management on both the macro and the individual (patient) scale.

Figure 5: Edward Tenner’s excellent book, Why Things Bite Back explores many examples and a number of reasons why technological advances often fail to reach their expected potential, and in fact, not infrequently turn out to be self limiting, or even self defeating.

Some of the technological singularities just listed, vaccination, for instance, can have very serious practical, economic and societal consequences. Rapid and widespread introduction of vaccination into equatorial Africa by Christian missionaries, absent the concurrent introduction of agricultural and other infrastructure, resulted in a population explosion and mass famine which has not abated to this day. Oral contraception has resulted in huge demographic and social changes occurring within a single human generation; a heretofore unprecedented event in the history of our species.

While medical advances are usually perceived as an unalloyed good for the patients who will benefit from them, this is rarely, if ever the case. The discovery of x-rays opened the interior of the human body to non-invasive examination, but it also exposed the patients so viewed to initially unsuspected exposure to damaging radiation – a problem that persists in radiologic medicine through the present. Beyond the problem of unforeseen or unknown dangers, there is also the problem of technological bite back, or what Edward Tenner has called the “revenge of unintended consequences.”(7) This is a major adverse effect of technological singularities, and one which often robs them of much of their anticipated bounty – not just for societies, but for individuals as well.

As I’ve just pointed out,  new medical technologies are sharply constrained in their utility at their start due to our inexperience with their bite back potential, and with the possibility of unknown  direct adverse affects of the technology  itself. However, every great once in awhile there are peculiar exceptions, and it just so happens that cryonicists are ideally positioned to enjoy just such an exception, starting now.

References

1. Fasouliotis, Sozos J, Schenker, Joseph G, TI, Cryopreservation of embryos: Medical, ethical, and legal issues. Journal of Assisted Reproduction and Genetics. 13:10 56-76;1996.

2. Simmons , RG, Fulton , J, Fulton, RF. The Prospective Organ Transplant Donor: Problems and Prospects of Medical Innovation. OMEGA–Journal of Death and Dying. 3:4;319-339:1972

3. Carrell. JL, The Speckled Monster: A Historical Tale of Battling the Smallpox Epidemic, Dutton, 2003, ISBN-10: 0525947361.

4. McKinlay, Kantor, If The South Had Won The Civil War, Forge Books, 2001, ISBN-10: 0312869495.

5. Murphy, FP, “Ignaz Philipp Semmelweis (1818–1865): An Annotated Bibliography,” Bulletin of the History of Medicine 20(1946), 653-707: 654f.

6. Herr, HWJ, Ignorance is bliss: the Listerian revolution and  the education of American surgeons. Urology;177:457-60,2007.

7. Tenner, EW, Why Things Bite Back: The Revenge of Unintended Consequences, Vintage, 1997, ISBN-10: 0679747567.

I.    Introduction                                  

II.   Materials and Methods

III   Effects of Glycerolization

IV.  Gross Effects of Cooling to and Rewarming From -196°C

I. INTRODUCTION

The  immediate  goal  of human cryopreservation  is  to  use  current cryobiological  techniques  to  preserve the  brain  structures  which encode personal identity adequately enough to allow for  resuscitation or reconstruction of the individual should molecular nanotechnology be realized (1,2).  Aside from two previous isolated efforts (3,4)  there has  been  virtually no systematic effort to examine the  fidelity  of histological,  ultrastructural, or even gross structural  preservation of  the brain following cryopreservation in either an animal or  human model.    While  there  is  a  substantial  amount  of  indirect   and fragmentary  evidence  in the  cryobiological  literature  documenting varying  degrees  of  structural  preservation  in  a  wide  range  of mammalian tissues (5,6,7), there is little data of direct relevance to cryonics.   In particular, the focus of contemporary  cryobiology  has been   on   developing  cryopreservation  techniques   for   currently transplantable  organs,  and this has necessarily  excluded  extensive cryobiological  investigation  of  the brain, the  organ  of  critical importance to human identity and mentation.

The  principal  objective of this pilot study was to  survey  the effects of glycerolization, freezing to liquid nitrogen  temperature, and  rewarming  on  the physiology, gross  structure,  histology,  and ultrastructure of both the ischemic and non-ischemic adult cats  using a preparation protocol similar to the one then in use on human cryopreservation patients.  The non-ischemic group was given the designation Feline Glycerol Perfusion (FGP) and the ischemic group was referred to as Feline Ischemic Glycerol Perfusion (FIGP).

The work described in this paper was carried out over a  19-month period from January, 1982 through July, 1983.  The perfusate  employed in this study was one which was being used in human cryopreservation operations at that time, the composition of which is given in Table I.

The principal cryoprotectant was glycerol.

II. MATERIALS AND METHODS

Pre-perfusion Procedures

Nine adult cats weighing between 3.4 and 6.0 kg were used in this study.  The animals were divided evenly into a non-ischemic and a  24-hour mixed warm/cold ischemic group.  All animals received humane care in  compliance  with  the  “Principles  of  Laboratory  Animal   Care” formulated by the National Society for Medical Research and the “Guide for  the Care and Use of Laboratory Animals” prepared by the  National Institutes  of  Health  (NIH Publication  No.  80-23,  revised  1978).  Anesthesia   in  both  groups  was  secured  by  the   intraperitoneal administration of 40 mg/kg of sodium pentobarbital.  The animals  were then  intubated and placed on a pressure-cycled ventilator.   The  EKG was monitored throughout the procedure until cardiac arrest  occurred. Rectal and esophageal temperatures were continuously monitored  during perfusion using YSI type 401 thermistor probes.

Following placement of temperature probes, an IV was  established in  the medial foreleg vein and a drip of Lactated Ringer’s was  begun to  maintain  the  patency of the IV and  support  circulating  volume during  surgery. Premedication (prior to perfusion) consisted of  the IV  administration of 1 mg/kg of metubine iodide to inhibit  shivering during  external  and  extracorporeal cooling  and  420  IU/kg  sodium heparin  as  an anticoagulant.  Two 0.77 mm I.D.  Argyle  Medicut  15″ Sentinel line catheters with Pharmaseal K-69 stopcocks attached to the luer fittings of the catheters were placed in the right femoral artery and vein.  The catheters were connected to Gould Model P23Db  pressure transducers   and  arterial  and  venous  pressures   were   monitored throughout the course of perfusion.

Surgical Protocol

Following placement of the monitoring catheters, the animals were transferred  to a tub of crushed ice and positioned for surgery.   The chest  was shaved and a median sternotomy was performed.   The  aortic root was cleared of fat and a purse-string suture was placed,  through which  a  14-gauge  Angiocath was introduced.   The  Angiocath,  which served  as  the  arterial  perfusion cannula,  was  snared  in  place, connected  to  the  extracorporeal circuit and cleared  of  air.   The pericardium  was  opened  and tented to expose the  right  atrium.   A purse-string  suture was placed in the apex of the right atrium and  a USCI  type  1967 16 fr. venous cannula was introduced  and  snared  in place.  Back-ties were used on both the arterial and venous cannulae to secure  them and prevent accidental dislodgment during the  course  of perfusion.  Placement of cannulae is shown in Figure 1.

Figure 1: Vascular access for extracorporeal perfusion was via median sternotomy. The arterial cannula consisted of a 14-gauge  Angiocath (AC) which was placed in the aortic root (AR) and secured in place with a purse string suture. A USCI  type  1967 16 fr. venous cannula (VC) was placed in the right atrium (RA) and snared in place using 0-silk ligature and a length of Red Robinson urinary catheter (snare). The chest wound was kept open using a Weitlander retractor. The left ventricle (LV) was not vented.

  Extracorporeal Circuit

Figure 2: Cryoprotective perfusion apparatus: RR = recirculating reservoir, PMC = arterial pressure monitor and controller, MBD = micro-bubble detector, US = ultrasonic sensor, ADC = arterial drip chamber, D/0 = dialyzer/oxygenator, RP = cryoprotective ramp pump, HEX = arterial heat exchanger, 40 MFH = 40 micron filter holder, PT = arterial pressure transducer, CR = glycerol concentrate reservoir, EKG = electrocardiograph, TT = thermistor thermometer, TS = thermistor switch box, IB = ice bath, EC = electrocautery, APD = arterial pressure display.

The extracorporeal circuit (Figures 2&3) was of composed of 1/4″ and 3/8″  medical grade polyvinyl chloride tubing.  The circuit  consisted of  two  sections:  a  recirculating loop  to  which  the  animal  was connected  and a glycerol addition system.  The  recirculating  system consisted  of  a  10 liter polyethylene reservoir  positioned  atop  a magnetic  stirrer, an arterial (recirculating) roller pump,  an  Erika HPF-200  hemodialyzer which was used as a hollow fiber oxygenator  (8) (or alternatively, a Sci-Med Kolobow membrane oxygenator), a  Travenol Miniprime  pediatric  heat  exchanger, and a 40-micron  Pall  LP  1440 pediatric blood filter.  The recirculating reservoir was  continuously stirred with a 2″ Teflon-coated magnetic stir bar driven by a  Corning PC  353 magnetic stirrer.  Temperature was continuously  monitored  in the  arterial line approximately 15.2 cm from the arterial  cannula using a Sarns in-line thermistor temperature probe and YSI 42SL remote sensing  thermometer.  Glycerol concentrate was continuously added  to the recirculating system using a Drake-Willock dual raceway hemodialysis pump, while venous perfusate was concurrently withdrawn from the circuit and discarded using a second raceway in the same pump head.

Figure 3: Schematic of cryoprotective perfusion circuit.

Storage and Reuse of the Extracorporeal Circuit

After  use the circuit was flushed extensively with filtered  tap and distilled water, and then flushed and filled with 3%  formaldehyde in distilled water to prevent bacterial overgrowth.  Prior to use  the circuit was again thoroughly flushed with filtered tap water, and then with  filtered distilled water (including both blood and gas sides  of the hollow fiber dialyzer; Kolobow oxygenators were not re-used).   At the  end  of  the distilled water flush, a test for  the  presence  of residual formaldehyde was performed using Schiff’s Reagent.  Prior  to loading  of  the perfusate, the circuit was rinsed with 10  liters  of clinical  grade normal saline to remove any particulates  and  prevent osmotic dilution of the base perfusate.

Pall filters and arterial cannula were not re-used.  The  circuit was replaced after a maximum of three uses.

Preparation of Control Animals

Fixative Perfusion

Two control animals were prepared as per the above.  However, the animals  were subjected to fixation after induction of anesthesia  and placement  of cannulae.  Fixation was achieved by first perfusing  the animals   with  500  mL  of  bicarbonate-buffered  Lactated   Ringer’s containing 50 g/l hydroxyethyl starch (HES) with an average  molecular weight  of  400,000 to 500,000 supplied by  McGaw  Pharmaceuticals  of Irvine, Ca (pH adjusted to 7.4) to displace blood and facilitate  good distribution of fixative, followed immediately by perfusion of 1 liter of  modified  Karnovsky’s  fixative (Composition given  in  Table  I).  Buffered Ringers-HES perfusate and Karnovsky’s solution were  filtered through 0.2 micron filters and delivered with the same  extracorporeal circuit described above.

Immediately   following  fixative  perfusion  the  animals   were dissected and 4-5 mm thick coronal sections of organs were cut, placed in  glass screw-cap bottles, and transported, as detailed  below,  for light or electron microscopy.

Straight Frozen Non-ischemic Control

One animal was subjected to straight freezing (i.e., not  treated with   cryoprotectant).    Following  induction  of   anesthesia   and intubation  the  animal  was supported on  a  ventilator  while  being externally  cooled  in  a  crushed  ice-water  bath.   When  the   EKG documented  profound bradycardia at 26°C, the animal was  disconnected from  the  ventilator,  placed  in a  plastic  bag,  submerged  in  an isopropanol  cooling bath at -10°C, and chilled to dry ice and  liquid nitrogen  temperature  per the same protocol used for  the  other  two experimental groups as described below.

Preparation of FGP Animals

Following  placement of cannulae, FGP animals were  subjected  to total  body  washout  (TBW) by open-circuit perfusion  of  500  mL  of glycerol-free  perfusate.  The extracorporeal circuit was then  closed and constant-rate addition of glycerol-containing perfusate was begun.

Cryoprotective  perfusion continued until the target concentration  of glycerol  was reached or the supply of glycerol-concentrate  perfusate was exhausted.

Preparation of FIGP Animals

In   the  FIGP  animals,  ventilator  support  was   discontinued following anesthesia and administration of Metubine.  The endotracheal tube was clamped and the ischemic episode was considered to have begun when cardiac arrest was documented by absent EKG.

After the start of the ischemic episode the animals were  allowed to  remain on the operating table at room temperature ( 22°C to  25°C) for  a  30  minute period to simulate  the  typical  interval  between pronouncement  of legal death in a clinical environment and the  start  of  external cooling at that time.  During the 30 minute  normothermic ischemic  interval the femoral cut-down was performed  and  monitoring lines were placed in the right femoral artery and vein as per the  FGP animals.  Prior to placement, the monitoring catheters were  irrigated with normal saline, and following placement the catheters were  filled with 1000 unit/mL of sodium heparin to guard against clot  obstruction of the catheter during the post-arrest ischemic period.

Figure 4: Typical cooling curve of FIGP animals to ~1°C following cardiac arrest.

After the 30 minute normothermic ischemic period the animals were placed  in  a  1-mil polyethylene bag,  transferred  to  an  insulated container  in  which  a bed of crushed ice had  been  laid  down,  and covered  over with ice.  A typical cooling curve for a FIGP animal  is presented in Figure 4. FIGP animals were stored on ice in this fashion for a period of 24 hours, after which time they were removed from  the container and prepared for perfusion using the surgical and  perfusion protocol described above.

Perfusate

 TABLE I

Perfusate components were reagent or USP grade and were dissolved in USP grade water for injection.  Perfusate was pre-filtered through a Whatman GFB glass filter (a necessary step to remove precipitate)  and then passed through a Pall 0.2 micron filter prior to loading into the extracorporeal circuit.

Perfusion

Perfusion  of both groups of animals was begun by carrying out  a total body washout (TBW) with the base perfusate in the absence of any cryoprotective agent.  In the FGP group washout was achieved within  2 –  3 minutes of the start of open circuit asanguineous perfusion at  a flow rate of 160 to 200 mL/min and an average perfusion pressure of 40 mm Hg.   TBW  in  the  FGP  group  was  considered  complete  when  the hematocrit  was  unreadable and the venous effluent was  clear.   This typically was achieved after perfusion of 500 mL of perfusate.

Complete blood washout in the FIGP group was virtually impossible to  achieve (see “Results” below).  A decision was made prior  to  the start  of  this  study (based on  previous  clinical  experience  with ischemic human cryopreservation patients) not to allow the  arterial pressure  to  exceed  60  mm Hg for any  significant  period  of  time.  Consequently, peak flow rates obtained during both total body  washout and subsequent glycerol perfusion in the FIGP group were in the  range of 50-60 mL/min at a mean arterial pressure of 50 mm Hg.

Due to the presence of massive intravascular clotting in the FIGP animals  it  was necessary to delay placement of the  atrial  (venous) cannula (lest the drainage holes become plugged with clots) until  the large  clots present in the right heart and the superior and  inferior vena  cava  had been expressed through the atriotomy.  The  chest  was kept  relatively  clear of fluid/clots by active suction  during  this interval.   Removal  of  large clots and reasonable  clearing  of  the effluent  was usually achieved in the FIGP group after 15  minutes  of open  circuit asanguineous perfusion, following which the circuit  was closed and the introduction of glycerol was begun.

Figure 5: pH of non-ischemic Δ•▪*(FGP) and ischemic ●●● ᴑ  (FIGP) cats during cryoprotective perfusion. The FIGP animals were, as expected, profoundly acidotic with the initial arterial pH being between 6.5 and 6.6.

The  arterial pO₂ of animals in both the FGP and FIGP groups  was kept  between  600  mm Hg and 760 mm Hg throughout  TBW  and  subsequent glycerol  perfusion.  Arterial pH in the FGP animals was  between  7.1 and  7.7  and was largely a function of the degree of  diligence  with which  addition of buffer was pursued.  Arterial pH in the FIGP  group was 6.5 to 7.3.  Two of the FIGP animals were not subjected to  active buffering during perfusion and as a consequence recovery of pH to more normal  values  from the acidosis of ischemia (starting  pH  for  FIGP animals was typically 6.5 to 6.6) was not as pronounced (Figure 5).

Figure 6: Calculated versus actual increase in arterial and venous glycerol concentration in the FGP animals. Arrow indicates actual time of termination of perfusion.

Introduction  of glycerol was by constant rate addition  of  base perfusate  formulation  made up with 6M glycerol  to  a  recirculating reservoir  containing 3 liters of glycerol-free base  perfusate.   The target  terminal tissue glycerol concentration was 3M and  the  target time  course for introduction was 2 hours.  The volume of 6M  glycerol concentrate  required  to  reach  a  terminal  concentration  in   the recirculating   system  (and  thus  presumably  in  the  animal)   was calculated as follows:

Vp

Mc = ——— Mp

Vc + Vp

where

Mc = Molarity of glycerol in animal and circuit.

Mp = Molarity of glycerol concentrate.

Vc = Volume of circuit and exchangeable volume of animal.*

Vp = Volume of perfusate added.

* Assumes an exchangeable water volume of 60% of the pre-perfusion  weight of the animal.

Glycerolization  of  the FGP animals was carried out at  10°C  to 12°C.   Initial  perfusion  of FIGP animals was at  4°C  to  5°C  with warming  (facilitated  by  TBW with warmer perfusate  and  removal  of surface  ice packs) to 10°-12°C for cryoprotectant introduction.   The lower  TBW  temperature of the FIGP animals was a consequence  of  the animals  having  been refrigerated on ice for the 24  hours  preceding perfusion.

Following  termination  of the cryoprotective ramp,  the  animals were  removed  from bypass, the aortic cannula was left  in  place  to facilitate  prompt reperfusion upon rewarming, and the venous  cannula was removed and the right atrium closed.  The chest wound was  loosely closed using surgical staples.

Concurrent with closure of the chest wound, a burr hole craniotomy 3  to  5  mm in diameter was made in the right parietal  bone  of  all animals  using a high speed Dremel “hobby” drill.  The purpose of  the burr hole  was  to  allow for  post-perfusion  evaluation  of  cerebralvolume, assess the degree of blood washout in the ischemic animals and facilitate  rapid expansion of the burr hole on re-warming to allow  for the visual evaluation of post-thaw reperfusion (using dye).

The  rectal  thermistor probe used to  monitor  core  temperature during  perfusion was replaced by a copper/constantan thermocouple  at the  conclusion  of perfusion for monitoring of the  core  temperature during cooling to -79°C and -196°C.

Cooling to -79°C

Figure 7: Representative cooling curve (esophageal and rectal temperatures) of FGP and FIGP animals from ~ 10°C to ~ -79°C. The ragged curve with sharp temperature excursions and rebounds is an artifact of the manual control of temperature descent via the addition of chunks of dry ice.

Cooling  to -79°C was carried out by placing the  animals  within two 1 mil polyethylene bags and submerging them in an isopropanol bath which  had  been  pre-cooled to -10°C.   Bath  temperature  was  slowly reduced  to  -79°C  by the periodic addition of dry  ice.   A  typical cooling curve obtained in this fashion is shown in Figure 7.   Cooling was at a rate of approximately 4°C per hour.

Cooling to and Storage at -196°C

Figure 8: Animals were cooled to -196°C by immersion in liquid nitrogen (LN₂) vapor in a Linde LR-40 cryogenic dewar. When a core temperature of ~-180 to -185°C was reached, the animals were immersed in LN₂.

Following cooling to -79°C, the plastic bags used to protect  the animals  from  alcohol were removed, the animals  were  placed  inside nylon  bags with draw-string closures and were then positioned atop  a 6″ high aluminum platform in an MVE TA-60 cryogenic dewar to which 2″-3″ of liquid nitrogen had been added.  Over a period of  approximately 15  hours  the liquid nitrogen level was gradually  raised  until  the animal  was  submerged.  A typical cooling curve  to  liquid  nitrogen temperature  for animals in this study is shown in Figure 8.   Cooling rates to liquid nitrogen temperature were approximately 0.178°C per  hour.  After  cool-down  animals  were maintained in liquid  nitrogen  for  a period  of  6-8  months until being removed  and  re-warmed  for  gross structural, histological, and ultrastructural evaluation.

Re-warming

Figure 9: Rewarming of all animals was accomplished by removing the animals from LN2 and placing them in a pre-cooled box insulated with 15.2 cm of polyurethane (isocyothianate) foam to which 1.5 L of LN2 (~2 cm on the bottom of the box)  of LN2 had been added. When the core temperature of the animals reached -20°C the animals were transferred to a mechanical refrigerator at 3.4°C.

The  animals  in  both groups were re-warmed to -2°C  to  -3°C  by removing them from liquid nitrogen and placing them in a pre-cooled box insulated on all sides with a 10.2 cm thickness of Styrofoam and containing a small quantity of liquid nitrogen.  The animals were then allowed to re-warm to approximately -20°C, at which time they were transferred  to a  mechanical  refrigerator at a temperature of 8°C.   When  the  core temperature  of the animals had reached -2°C to -3°C the animals  were removed to a bed of crushed ice for dissection, examination and tissue collection  for  light and electron microscopy.  A  typical  re-warming curve is presented in Figure 9.

Modification of Protocol Due To Tissue Fracturing

After the completion of the first phase of this study  (perfusion and  cooling  to  liquid nitrogen temperature)  the  authors  had  the opportunity  to evaluate the gross and histological condition  of  the remains  of three human cryopreservation patients who  were  removed from  cryogenic  storage  and  converted  to  neuropreservation  (thus allowing  for post-arrest dissection of the body, excluding the  head) (10).  The results of this study confirmed previous, preliminary, data indicative of gross fracturing of organs and tissues in animals cooled to  and  re-warmed from -196°C.  These findings led us to  abandon  our plans  to  reperfuse  the  animals  in  this  study  with  oxygenated, substrate-containing  perfusate  (to have been  followed  by  fixative perfusion  for histological and ultrastructural evaluation) which  was to be have been undertaken in an attempt to assess post-thaw viability by  evaluation  of post-thaw oxygen consumption, glucose  uptake,  and tissue-specific enzyme release.

Re-warming  and  examination  of the first  animal  in  the  study confirmed  the presence of gross fractures in all organ systems.   The scope  and severity of these fractures resulted in disruption  of  the circulatory system, thus precluding any attempt at reperfusion as  was originally planned.

Preparation of Tissue Samples For Microscopy

Fixation

 TABLE II.

Samples of four organs were collected for subsequent histological and  ultrastructural  examination:  brain, heart,  liver  and  kidney.  Dissection  to  obtain  the tissue samples was begun as  soon  as  the animals  were  transferred to crushed ice.  The brain  was  the  first  organ  removed  for sampling.  The burr hole created at  the  start  of perfusion  was  rapidly extended to a full craniotomy  using  rongeurs (Figure  14).   The  brain was then removed en bloc to  a  shallow  pan containing  iced,  modified Karnovsky’s fixative  containing  25%  w/v glycerol  (see  Table  II  for composition)  sufficient  to  cover  it.  Slicing of the brain into 5 mm thick sections was carried out with the brain  submerged  in fixative in this manner.  At  the  conclusion  of slicing  a 1 mm section of tissue was excised from the  visual  cortex and  fixed  in a separate container for electron  microscopy.   During final  sample  preparation for electron microscopy care was  taken  to avoid  the  cut  edges  of the tissue block  in  preparing  the  Epon embedded sections.

Figure 10: The sagitally sectioned (5 mm thickness) brains of the animals were placed in a  perforated basket immersed in Karnofsky’s fixative. This assembly was placed atop a magnetic stirring table and the fixative was gently  stirred with a magnetic stirring bar.

      The  sliced  brain  was  then placed in  350  ml  of  Karnovsky’s containing  25%w/v glycerol in a special stirring apparatus  which  is illustrated  in Figure 10.  This  fixation/de-glycerolization  apparatus consisted of two plastic containers nested inside of each other atop a magnetic stirrer.  The inner container was perforated with numerous  3 mm holes and acted to protect the brain slices from the stir bar which continuously  circulated the fixative over the slices.   The  stirring reduced  the likelihood of delayed or poor fixation due to overlap  of slices  or stable zones of tissue water stratification.   (The  latter was a very real possibility owing to the high viscosity of the  25%w/v glycerol-containing Karnovsky’s.)

De-glycerolization of Samples

Figure 11: Following fixation, the tissues slices of all organs evaluated by microscopy were serially de-glycerolized using the scheme shown above. When all of the glycerol was unloaded from the tissues they were shipped in modified Karnovsky’s to outside laboratories for histological and electron microscopic imaging.

          To avoid osmotic shock all tissue samples were initially immersed in Karnovsky’s containing 25%w/v glycerol at room temperature and were subsequently  de-glycerolized  prior  to  staining  and  embedding   by stepwise    incubation    in   Karnovsky’s    containing   decreasing concentrations  of  glycerol  (see  Figure  11  for the de-glycerolization protocol).

Figure 12: Fixation and de-glycerolization set up employed to prepare tissues for subsequent microscopic examination. Karnofsky’s fixative (A) was added to the tissue slice fixation apparatus (B) and the tissue slices were then subjected to serial immersion in fixative bathing media containing progressively lower concentrations of glycerol (C) (see Figure 11).

      To  prepare  tissue sections from heart, liver,  and  kidney  for microscopy,  the  organs  were  first removed  en  bloc  to  a  beaker containing an amount of ice-cold fixative containing 25% w/v  glycerol sufficient  to cover the organ.  The organ was then removed to a  room temperature  work  surface at where 0.5 mm sections were made  with  a Stadie-Riggs microtome.  The microtome and blade were pre-wetted  with fixative,  and cut sections were irrigated from the microtome  chamber into  a beaker containing 200 ml of room-temperature fixative using  a plastic  squeeze-type  laboratory  rinse  bottle  containing  fixative solution.   Sections  were  deglycerolized using  the  same  procedure previously detailed for the other slices.

Osmication and Further Processing

At  the  conclusion  of de-glycerolization of  the  specimens  all tissues  were  separated into two groups; tissues to be  evaluated  by light microscopy, and those to be examined with transmission  electron microscopy.   Tissues for light microscopy were shipped  in  glycerol-free  modified  Karnovsky’s solution to American  Histolabs,  Inc.  in Rockville,  MD  for  paraffin  embedding,  sectioning,  mounting,  and staining.

Tissues   for  electron  microscopy  were  transported   to   the facilities  of the University of California at San Diego in  glycerol-free  Karnovsky’s at 1° to 2°C for osmication, Epon embedding, and  EM preparation of micrographs by Dr. Paul Farnsworth.

Due  to  concerns  about the osmication and  preparation  of  the material processed for electron microscopy by Farnsworth, tissues from the  same  animals  were also submitted  for  electron  microscopy  to Electronucleonics of Silver Spring, Maryland.

III. EFFECTS OF GLYCEROLIZATION

 Perfusion of FGP Animals

Blood  washout  was  rapid and complete in the  FGP  animals  and vascular  resistance  decreased  markedly  following  blood   washout.  Vascular  resistance increased steadily as the glycerol  concentration increased,  probably  as a result of the increasing viscosity  of  the perfusate.

Within   approximately  5  minutes  of  the  beginning   of   the cryoprotective ramp, bilateral ocular flaccidity was noted in the  FGP animals.   As  the perfusion proceeded, ocular  flaccidity  progressed until  the  eyes had lost approximately 30% to 50%  of  their  volume.

Gross  examination  of the eyes revealed that initial water  loss  was primarily  from the aqueous humor, with more significant  losses  from the posterior chamber of the eyes apparently not occurring until later in  the  course  of  perfusion.  Within 15 minutes  of  the  start  of glycerolization  the corneal surface became dimpled and irregular  and the eyes had developed a “caved-in” appearance.

Dehydration  was also apparent in the skin and  skeletal  muscles and  was  evidenced  by  a marked decrease  in  limb  girth,  profound muscular  rigidity,  cutaneous  wrinkling (Figure 11),  and  a  “waxy-leathery” appearance and texture to both cut skin and skeletal muscle.

Tissue water evaluations conducted on ileum, kidney, liver, lung,  and skeletal  muscle  confirmed  and  extended  the  gross   observations.

Figure 13: Cutaneous dehydration following glycerol perfusion is evidenced by washboard wrinkling of the thoraco-abdominal skin (CD). The ruffled appearance of the fur on the right foreleg (RF) is also an artifact of cutaneous dehydration. The sternotomy wound, venous cannula and the Weitlaner retractor (R) and the retractor blade (RB) holding open the chest wound are visible at the upper left of the photo.

Preliminary  observation suggest that water loss was in the  range  of 30%  to 40% in most tissues. As can be seen in Table III,  total  body water  losses  attributable  to dehydration, while  typically  not  as profound, were still in the range of 18% to 34%.  The gross appearance of  the heart suggested a similar degree of dehydration, as  evidenced by modest shrinkage and the development of a “pebbly” surface  texture and a somewhat translucent or “waxy” appearance.

TABLE III.

Figure 14: Cerebrocortical dehydration as a result of 4M glycerol perfusion. The cortical surface (CS) is retracted ~5-8 mm below the margin of the cranial bone (CB).

Examination  of  the cerebral hemispheres through the  burr  hole (Figure  14) and of the brain in the brain brainpan (Figure 19) revealed an estimated 30% to 50% reduction  in  cerebral volume,  presumably  as a result of osmotic dehydration  secondary  to glycerolization.   The cortices also had the “waxy”  amber  appearance previously observed as characteristic of glycerolized brains.

The  gross  appearance  of the kidneys,  spleen,  mesenteric  and subcutaneous  fat, pancreas, and reproductive organs  (where  present) were   unremarkable.   The  ileum  and  mesentery  appeared   somewhat dehydrated,  but  did  not  exhibit  the  waxy  appearance  that   was characteristic of muscle, skin, and brain.

Figure 15: Oxygen consumption was not apparently affected by glycerolization as can be seen in the data above from the perfusions of FGP-5 and FGP-5.

Oxygen  consumption (determined by measuring the  arterial/venous difference)  throughout  perfusion  was fairly constant  and  did  not appear to be significantly impacted by glycerolization, as can be seen Figure 12.

Perfusion of FIGP Animals

As previously noted, the ischemic animals had far lower flow rates at  the  same  perfusion  pressure as  FGP  animals  and  demonstrated incomplete  blood  washout.   Intravascular  clotting  was  serious  a barrier  to  adequate perfusion.   Post-thaw  dissection  demonstrated multiple  infarcted areas in virtually all organ systems; areas  where blood  washout  and  glycerolization were incomplete  or  absent.   In contrast  to  the even color and texture changes observed in  the  FGP animals,  the  skin of the FIGP animals  developed  multiple,  patchy, non-perfused   areas  which  were  clearly  outlined  by   surrounding, dehydrated, amber-colored glycerolized areas.

External  and internal examination of the brain and  spinal  cord revealed  surprisingly  good  blood washout  of  the  central  nervous system.  While grossly visible infarcted areas were noted, these  were relatively  few  and  were generally no larger than 2 mm to  3  mm  in diameter.   With few exceptions, the pial vessels were free  of  blood and appeared empty of gross emboli.  One striking difference which was consistently  observed  in  FIGP  animals  was  a  far  less  profound reduction  in brain volume during glycerolization (Figure  17).   This may  have  been due to a number of factors: lower flow  rates,  higher perfusion  pressures,  and the increased  capillary  permeability  and perhaps increased cellular permeability to glycerol.

Figure 16: The eye of an FGP animal following cryopreservation. The cornea has  become concave due to the glycerol-induced osmotic evacuation of the aqueous humor. The vitreous humor is completely obscured by the lens which has become white and opaque as a result of the precipitation of the crystallin proteins in the lens.

Whereas   edema   was   virtually   never   a   problem    during glycerolization  of  FGP  animals, edema was  universal  in  the  FIGP animals  after as little as 30 minutes of perfusion.  In  the  central nervous  system this edema was evidenced by a “rebound”  from  initial cerebral  shrinkage  to  frank  cerebral  edema,  with  the  cortices, restrained by the dura, often abutting or slightly projecting into the burr hole.   Marked  edema of the nictating membranes,  the  lung,  the intestines,  and  the  pancreas  was also a  uniform  finding  at  the conclusion  of cryoprotective perfusion.  The development of edema  in the central nervous system sometimes closely paralleled the  beginning of “rebound” of ocular volume and the development of ocular turgor and frank ocular edema.

Figure 17: The appearance of the brain of an FIGP animal following cryoprotective perfusion as seen through a craniotomy performed over the right temporal lobe. The cortical surface (CS) is retracted ~3-5 mm from the cranial bone (CB) and appears

In contrast to the relatively good blood washout observed in  the brain,  the  kidneys  of  FIGP animals had a  very  dark  and  mottled appearance.   While  some  areas (an estimated  20%  of  the  cortical surface) appeared to be blood-free, most of the organ remained  blood-filled throughout perfusion.  Smears of vascular fluid made from renal biopsies  which  were collected at the conclusion  of  perfusion  (for tissue  water determinations) revealed the presence of many  free  and irregularly clumped groups of crenated and normal-appearing red cells, further evidence of the incompleteness of blood washout.   Microscopic examination  of recirculating perfusate revealed some free, and a  few clumped  red  cells.   However, the concentration  was  low,  and  the perfusate  microhematocrit  was  unreadable  at  the  termination   of perfusion (i.e., less than 1%).

The  liver  of  FIGP  animals  appeared  uniformly   blood-filled throughout  perfusion,  and  did not exhibit even  the  partial  blood washout evidenced by the kidneys.  However, despite the absence of any grossly  apparent blood washout, tissue water evaluations in one  FIGP animal  were  indicative  of  osmotic dehydration  and  thus  of  some perfusion.

The mesenteric, pancreatic, splanchnic, and other small  abdominal vessels  were  largely free of blood by the conclusion  of  perfusion.  However,  blood-filled  vessels  were not  uncommon,  and  examination during   perfusion   of   mesenteric   vessels   performed   with   an ophthalmoscope  at 20x magnification revealed stasis in  many  smaller vessels, and irregularly shaped small clots or agglutinated masses  of red  cells in most of the mesenteric vessels.   Nevertheless,  despite the   presence  of  massive  intravascular  clotting,  perfusion   was possible, and significant amounts of tissue water appear to have  been exchanged for glycerol.

One  immediately  apparent difference between the  FGP  and  FIGP animals  was  the  accumulation in the lumen of  the  ileum  of  large amounts  of  perfusate  or perfusate  ultrafiltrate  by  the  ischemic animals.  Within approximately 10 minutes of the start of reperfusion, the  ileum  of the ischemic animals that had  been  laparotomized  was noticed  to  be  accumulating fluid.  By the  end  of  perfusion,  the stomach  and the small and large bowel had become massively  distended with  perfusate.   Figure  14 shows both FIGP and  FGP  ileum  at  the conclusion  of glycerol perfusion.  As can be clearly seen,  the  FIGP intestine  is markedly distended.  Gross examination of the  gut  wall was   indicative  of  tissue-wall  edema  as  well   as   intraluminal accumulation  of  fluid.  Often by the end of perfusion, the  gut  had become  so  edematous  and  distended  with  perfusate  that  it   was impossible  to completely close the laparotomy  incision.   Similarly, gross  examination of gastric mucosa revealed severe erosion with  the mucosa being very friable and frankly hemorrhagic.

Escape  of  perfusate/stomach contents from the  mouth  (purging) which occurs during perfusion in ischemically injured human suspension patients did not occur, perhaps due to greater post-arrest  competence of the gastroesophageal valve in the cat.

Oxygen  consumption  in  the two ischemic cats in  which  it  was measured  was dramatically impacted, being only 30% to 50% of  control and deteriorating throughout the course of perfusion (Figure 12).

IV. GROSS EFFECTS OF COOLING TO AND REWARMING FROM -196°C

The  most striking change noted upon thawing of the  animals  was the presence of multiple fractures in all organ systems.  As had  been previously noted in human cryopreservation patients, fracturing  was most pronounced in delicate, high flow organs which are poorly  fiber-reinforced.   An exception to this was the large arteries such as  the aorta, which were heavily fractured.

Fractures  were most serious in the brain, spleen, pancreas,  and kidney.   In these organs fractures would often completely  divide  or sever  the  organ  into one or more discrete  pieces.   Tougher,  more fiber-reinforced tissues such as myocardium, skeletal muscle, and skin were less affected by fracturing; there were fewer fractures and  they were smaller and less frequently penetrated the full thickness of  the organ.

Figure 18: All of the animals in the study exhibited fractures of the white matter that transected the brain between the cerebellum and the cerebral cortices. Similarly, the spinal cord was invariable severed by fractures in several locations and exhibited the appearance of a broken candle stick. The yellow box encloses a sampling area used to determine brain water content.

Figure 19: Deep fracture of the left occipital cortex. Note the absence oif fracturing in the adjacent skeletal muscle (M) observed in FGP-1. Note that the brain appears shrunken and retracted in the brainpan.

Figure 20: Appearance of the brain after removal from the brainpan. There is a massive fracture of thew right frontal=temporal cortex which penetrates the full thickness of the cerebral hemisphere to expose the right cerebral ventricle observed in FIGP-2. The cortex appears buff colored and gives the appearance of being incompletely washed out of blood.

Figure 21: Typical fracture sites in the brain (arrows and yellow shading). The olfactory cortices and the brainstem were invariably completely severed by fractures.

In both FGP and FIGP animals the brain was particularly  affected by  fracturing  (Figures 18, 19 & 22) and  it  was not uncommon to  find  fractures  in  the cerebral hemispheres penetrating through to the ventricles as seen  in Figure  20, or to find most of both cerebral hemispheres and the  mid-brain  completely  severed from the cerebellum by a  fracture  (Figure 18).  Similarly, the cerebellum was uniformly severed from the medulla at the foramen magnum as were the olfactory lobes, which were  usually retained  within  the olfactory fossa with severing  fractures  having occurred at about the level of the transverse ridge.  The spinal  cord was  invariably transversely fractured at intervals of 5 mm to  15  mm over  its  entire  length (Figure 21).  Bisecting CNS fractures  were  most  often observed  to  occur  transversely  rather  than  longitudinally.   In general,  roughly  cylindrical structures such as  arteries,  cerebral hemispheres, spinal cord, lungs, and so on are completely severed only by transverse fractures.  Longitudinal fractures tend to be shorter in length and shallower in depth, although there were numerous exceptions to this generalization.

Figure 22: Crisp olfactory lobe fracture which also partially penetrated the pia matter in FGG-4.

In  ischemic animals the kidney was usually grossly fractured  in one  or  two locations (Figure 25).  By  contrast,  the  well-perfused kidneys of the non-ischemic FGP group exhibited multiple fractures,  as can  be  seen in Figure 24.  A similar pattern was observed  in  other organ  systems  as well; the non-ischemic animals  experienced  greater fracturing injury than the ischemic animals, presumably as a result of the   higher   terminal  glycerol  concentrations  achieved   in   the non-ischemic group.

Figure 23: Appearance of a fractured kidney before removal of the renal capsule. The renal capsule has only one fracture, however when the capsule is removed, the extensive fracturing of the renal cortex and medulla become evident (Figure 24, below).

Figure 24: Fractured renal cortex from FGP-1 after removal of the renal capsule. The renal cortex is extensively fractured, the renal medulla slightly less so. Note the uniform, tan/light brown color of the cortex indicating complete blood washout and the absence of red cell trapping.

Cannulae  and attached stopcocks where they were externalized  on the  animals  were  also frequently  fractured.   In  particular,  the polyethylene pressure-monitoring catheters were usually fractured into many  small  pieces.   The  extensive  fracture  damage  occurring  in cannulae,  stopcocks, and catheters was almost certainly a  result  of handling  the animals after cooling to deep subzero  temperatures,  as this  kind of fracturing was not observed in these items upon  cooling to  liquid nitrogen temperature (even at moderate rates).  It is  also possible that repeated transfer of the animals after cooling to liquid nitrogen  temperature may have contributed to fracturing  of  tissues, although the occurrence of fractures in organs and bulk quantities  of water-cryoprotectant  solutions  in the absence of  handling  is  well documented in the literature (12, 13).

There were subtle post-thaw alterations in the appearance of  the tissues of all three groups of animals.  There was little if any fluid present  in the vasculature and yet the tissues exhibited  oozing  and “drip”  (similar to that observed in the muscle of frozen-thawed  meat and  seafood)  when cut.  This was most pronounced  in  the  straight-frozen  animal.  The tissues (especially in the ischemic  group)  also had  a somewhat pulpy texture on handling as contrasted with  that  of unfrozen,  glycerolized  tissues  (i.e.,  those  handled  during  pre-freezing  sampling for water content).  This was most in  evidence  by the accumulation during the course of dissection of small particles of what appeared to be tissue substance with a starchy appearance and  an oily  texture on gloves and instruments .  This phenomenon  was  never observed  when handling fresh tissue or glycerolized tissue  prior  to freezing and thawing.

There were marked differences in the color of the tissues between the three groups of animals as well.  This was most pronounced in  the straight-frozen  control  where the color of almost  every  organ  and tissue examined had undergone change.  Typically the color of  tissues in  the  straight-frozen animal was darker, and white  or  translucent tissues such as the brain or mesentery were discolored with hemoglobin released from lysed red cells.

Figure 25: The (ventral) dependent and dorsal (less dependent) surfaces of the right kidney from FIGP-1. There is extensive mottling evidencing incomplete blood washout despite perfusion with many liters of CPA solution. Fracturing is much less extensive than that observed in FGP animals not subjected to prolonged periods of post-arrest ischemia. Note the pink colored “drip” from the organ that is present on sectioning board.

Figure 26: Appearance of the kidney from FIGP-1 shown above on cross-section. The renal medulla appears congested and blood filled.

The FGP and FIGP groups did not experience the profound post-thaw changes  in tissue color experienced by the straight-frozen  controls, although  the  livers and kidneys of the FIGP  animals  appeared  very dark, even when contrasted with their pre-perfusion color as  observed in those animals laparotomized for tissue water evaluation.

END OF PART 1