The RhinoChill: A New Way to Cool the Brain Quickly

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

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

Read the complete article in PDF here.

At last, a sure-cold way to sell cryonics with guaranteed success!

A humorous romp through a promising new technique in aesthetic medicine from one cryonicist’s (warped) point of view.

Figure 1: Before cryopreservation (L) and after cryopreservation (R).

As everyone involved in cryonics for more than a fortnight is sadly aware, cryonics doesn’t sell. Indeed, if we were pitching a poke in the eye with a sharp stick, we’d more than likely have more takers than we’ve had trying to ‘market’ cryonics to the public. To see evidence that this is so, you need only wander around a shopping mall on a weekend and observe all the (painfully) stainless steel lacerated and brightly colored needle-pierced flesh sported by the young and trendy and increasing by the old and worn, as well.

Yes, it’s clear; we misread the market, to our lasting detriment.

It’s true that we’ve tried the ‘you’ll be rich when you wake you up line,’ and heaven knows we’ve beaten the ‘you’ll be young and beautiful forever’ line, well, virtually beaten it to death. And while people are certainly interested in great fortune and youth, both of these things share the same unfortunate shortcoming, namely that they are things that people either don’t have but want, or do have and don’t want to lose. As anyone who is really savvy at marketing will tell you, the best way to sell something is to promise (and preferably be able to deliver) that you can get rid of something that people have and really don’t want – something that is ruining the quality of their life, destroying their health, draining their pocketbook and, worst of all, making them really, really ugly.

So, it turns out that for onto 50 years now, we’ve missed the real selling point of cryonics that’s been there all along: IT WILL MAKE YOU THIN! Guaranteed!

Can such a claim be true? Well, surprisingly, the answer would seem to be an almost unqualified, “Yes!”

Recently it’s been discovered that adipocytes, the cells responsible not only for making you fat, but for making you hungry, as well, are particularly susceptible to a phenomenon in cryobiology that has proved a nettlesome (and only recently (partially) overcome) barrier to solid organ cryopreservation: chilling injury. Quite apart from freezing damage due to ice crystals forming, adipocytes are selectively vulnerable to something called ‘chilling injury.’ 1-5 Chilling injury occurs when tissues are cooled to a temperature where the saturated fats that comprise their cell membranes (external and internal) freeze. You see, saturated fat, which is the predominant type of fat in us humans, freezes well above the temperature of water – in fact, it freezes at just below room temperature. That’s why that big gash of fat on the edge of your T-bone steak is stiff and waxy when it is simply refrigerated, and not frozen.

Figure 2: Chilling injury is thought to result from crystallization of cell membrane lipids.

Chilling injury isn’t really well understood. In the days before both cryobiology and indoor heating, humans used to experience a very painful manifestation of it in the form of chilblains – tender swelling and inflammation of the skin due to prolonged cold exposure (without freezing haven taken place). In the realm of organ preservation it is currently thought that chilling injury occurs when cell membranes are exposed to high subzero temperatures (-5oC to -20oC), again, in the absence of freezing.

There is evidence that the lipids (fats) that make up the smooth, lamellar cell membranes undergo crystallization when cells are cooled much below 0 deg C. Since the crystals are hexagonal in shape and have a hole in the middle, this has the effect of creating a pore or hole in the membrane. Cells don’t like that – those holes let all kinds of ions important to cells keeping their proper volume and carrying on their proper metabolic functions leak in and out, as the case may be. This isn’t merely an inconvenience for cells, it’s downright lethal. Without boring you with technical details, it is possible to partially address this state of affairs in organ preservation by adjusting the ‘tonicity’ of the solution bathing the cells: oversimplifying even more, this means by increasing  the concentration of salts to a concentration higher than would normally be present

Figure 3: Contouring of the skin in a pig subjected to brief, subzero cooling of subcutaneous fat.

But, to return to our chilled adipocytes and the promise not only of weight loss, but of a fat-free future; adipocytes are killed, en masse, when their temperature is dropped to between 0 and -7oC. Within a few days of exposure to such temperatures they undergo programmed cell death (apoptosis) and within a couple of months they are phagocytized by the body; and all that ugly and unwanted fat is carted off to be used as fuel by the liver. Now the rub would seem to be that this effect is most pronounced when the temperature of the tissue is cooled to below the freezing point of water and held there – preferably for a period of 10 minutes or longer.

That sounds dire, doesn’t it? What about the skin, the fascia, blood vessels, and the other subcutaneous tissues that will FREEZE (in the very conventional sense of having lots and lots of ice form in them)? Well, the answer, as any long-time experimental cryobiologist will know (even if he won’t tell you) is: pretty much nothing. Way back in the middle of the previous century, a scientist named Audrey Smith and her colleagues at Mill Hill, England found that you could freeze hamsters ‘solid’ – freeze 70+% of the water in their skin and 50% of the water in their bodies – and they would recover from this procedure none the worse for wear. Similarly, those of us who have carelessly handled dry ice for a good part of our lives will tell you that we see parts of our fingertips turn into stiff chalky islands of ice all the time, with the only side effect being a bit of temporary numbness that resolves in a few days to a week – certainly a side effect well worth it to avoid the considerable inconvenience of rummaging around to find a pair of protective gloves.

Figure 4: The Zeltiq Cool Sculpting Cryolipolysis device.

But alas, we scientists (most of us, anyway) are not a very entrepreneurial lot, and so we never thought either of inventing the ZeltiqTM cryolipolysis system, or using ‘the thin-new-you’ as a marketing tool for cryonics.

Yes, that’s right; some very clever folks have found a way to make a huge asset out of a colossal liability – to organ preservationists, anyway. Around 2004 a Minneapolis dermatologist named Brian Zellickson, MD, who specialized in laser and ultrasonic skin rejuvenating procedures, made a not so obvious connection. Both laser and skin ‘face-lifting’ and skin ‘rejuvenation’ procedures rely on the subcutaneous delivery of injuring thermal energy to the tissues of the face, or other treated parts of the body (cellulite of the buttocks and thighs are two other common areas for treatment). These energy sources actually inflict a second degree burn in a patchy and well defined way to the subdermal tissues.

Now this may seem a very counterintuitive thing to do if you are trying to induce ‘rejuvenation’ or ‘lift’ a sagging face. But if you think about it, it makes a great deal of sense. As any burn victim will tell you, one of the most difficult (and painful) parts of recovery is stretching the highly contracted scar tissue that has formed as a result of the burn injury. Indeed, for many patients with serious burns over much of their body, the waxy, rubbery and very constricting scar tissue prevents the return of normal movement, and can lock fingers and even limbs into a very limited range of motion. Many burn victims must do painful stretching exercises on a daily basis to avoid the return of this paralyzing skin (scar) contracture.

And it must be remembered that aged skin – even the skin of the very old – can still do one thing, despite the many abilities it has lost with age, and that thing is to form scar tissue in response to injury. Thus, laser and ultrasonic heating of normal (but aged) skin induces collagen proliferation and large-scale remodeling of the skin. For all the bad things said about scar tissue it is still a remarkable achievement in that it does constitute regenerated tissue. Regenerated tissue which does the minimum that normal skin must do to keep us alive: provide a durable covering that excludes microbial invasion, and prevents loss of body fluids. By injuring the tissue just below the complexly differentiated layer of the dermis (with its hair follicles, sweat glands and highly ordered pigmentation cells) much of the benefit of ‘scarring’ is obtained without the usual downsides.

The injured tissues respond by releasing collagen building cytokines as well as cytokines that result in angiogenesis (new blood vessel formation) and widespread tissue remodeling. And all that newly laid down collagen contracts over time, tightening and lifting the skin – and the face it is embedded in. These techniques may justly be considered much safer versions of the old fashioned chemical face peel, which could be quite effective at erasing wrinkles and achieving facial ‘rejuvenation,’ but was not titrateable and was occasionally highly unpredictable: every once in awhile the result was disastrous burning and accompanying long term scarring and disfiguration of the patient’s face.

St some point Dr. Zellickson seems to have realized that the selective vulnerability of adipocytes to chilling offered the perfect opportunity for a truly non-invasive approach to ‘liposuctioning’ by using the body’s own internal suctioning apparatuses, the phagocytes, to do the job with vastly greater elegance and panache than any surgeon with a trocar and a suction machine could ever hope to do. Thus was invented the Zeltiq Cool SculptTM cryolipolysis machine.6

Figure 5: The cooling head of the Zeltiq devive equipped with ultrasonic imaging equipment and a suction device to induce regional ischemia and hold the tissue against the cooling surface.

The beauty of cryolipolysis is that it is highly titrateable, seems never to result to in excessive injury to, or necrosis of the overlying skin, and yields a smooth and aesthetically pleasing result. Not unjustifiably for this reason it is marketed under the name Cool SculptingTM. The mechanics of the technique are the essence of simplicity. The desired area of superficial tissue to be remodeled is entrained by vacuum in a cooling head equipped with temperature sensors, an ultrasonic imaging device, and a mechanical vibrator. The tissue in the cooling head is sucked against a conductive surface (made evenly conductive by the application of a gel or gel-like dressing to the skin) where heat is extracted from it. The tissue is cooled to a temperature sufficient to induce apoptosis in the adipocytes, while at the same time leaving the overlying skin untouched. The depth of cooling/freezing is monitored by ultrasound imaging and controlled automatically by the Zeltiq device.  At the appropriate point in the cooling process the tissue is subjected to a 5 minute period of mechanical agitation (massage) which helps to exacerbate the chilling injury, perhaps by nucleating the unfrozen fat causing it to freeze.7 When the treatment is over, the device pages an attendant to return to the treatment room and remove it.

The tissue under vacuum is also made ischemic – blood ceases to flow, and this has the dual advantage of speeding the course of the treatment by preventing the blood borne delivery of unwanted heat – and more importantly, by making the cooling more uniform, predictable and reproducible. It also has the effect of superimposing ischemic injury on top of the chilling injury which is something that seems to enhance adipocyte apoptosis. The whole treatment, in terms of actual cooling time, takes about 60 minutes. In the pig work which served as the basis for the human clinical treatments, the duration of treatment was only 10 minutes: but the cooling temperature was also an ‘unnerving’ -7oC. The degree of temporary and fully reversible peripheral nerve damage (that temporary numbness us ‘dry ice handlers’ know so well) was more severe at this temperature, although it resolved in days to a week or two, without exception.

As previously noted, cryolipolysis causes apoptosis of adipocytes and this results in their subsequently being targeted by macrophages that engulf and digest them. This takes time, and immediately after treatment there are no visible changes in the subcutaneous fat. However, three days after treatment, there is microscopic evidence that an inflammatory process initiated by the apoptosis of the adipocytes is underway, as evidenced by an influx of inflammatory cells into the fat of the treated tissues. This inflammatory process matures between seven and fourteen days after treatment; and between fourteen and thirty days post-treatment, phagocytosis of lipids is well underway. Thirty days after treatment the inflammatory process has begun to decline, and by 60 days, the thickness of interlobular septa in the fat tissue has increased. This last effect is very important because it is weakness, or failure of the interlobular fat septae that is responsible for the ugly ‘cottage cheese’ bulging that is cellulite. Three months after the treatment you get the effect you see below on the ‘love handles’ of this fit, and otherwise trim fellow. Thus, it is fair to say that Cool SculptingTM is in no way a misnomer.

Figure 6: Art left is a healthy, fit young male who has persistent accumulation of fat in the form of ‘love handles’ that are resistant to diet and exercise and the same man 3 months after cryolipolysis.

Does cryolipolysis really work? The answer is that it works extremely well for regional remodeling or sculpting of adipose tissue – those pesky love handles, that belly bulge around the navel, that too plump bum, or those cellulite marred thighs. So far it has not been used to try and ablate large masses of fat – although there seems no reason, in principal, why this could not be done using invasive techniques such as pincushioning the fat pannus with chilling probes, as is done with cryoablation in prostate surgery. However, this would be invasive, vastly more expensive, and likely to result in serious side effects.

And that was one of the really interesting things about the research leading up to FDA approval of cryolipolysis: it seems to cause no perturbation in blood lipids, no disturbance of liver function (the organ that has to process all that suddenly available fat) and no global alterations in immune function. It seems to be safe and largely adverse effect free. There is some localized numbness (as is the case in freezing of skin resulting from handling dry ice) but it resolves without incident with a few weeks of the procedure.8

So, all of this makes me wonder, since human tissues tolerate ice formation and respond to it in much the same way as they do to laser or ultrasound ‘rejuvenation’ (depending upon the degree of damage) a logical question is, “would it be possible to use partial freezing of the skin – just enough to provoke the remodeling response – as a method of facial rejuvenation?” It should be safer than a chemical people and it is, like laser and ultrasound therapy, titrateable.

Figure 7: “Gad darn it, this shiny gold stuff keeps getting into the silt I’m tryin to git out of this here river!”

Which returns me to the whole subject of cryonics: fat is very poorly perfused and it seems unlikely that things done to moderate or abolish chilling injury will be nearly so effective for the adipocytes in fat (if it they are effective at all). That means that we might well all come back from our cryogenic naps not only young, via the magic of nanotechnology and stem cell medicine, and rich via the miracle of compound interest (which none other than Albert Einstein once remarked was “the most powerful force in the universe”), but also THIN! For all these years organ cryopreservationists, like Fahy and Wowk, have been panning for the mundane silt of a way around a chilling injury9 all the while discarding the gleaming nuggets of gold that were persistently clogging up their pans.

We cryonicists should not repeat their error and should realize a good thing when we see it. Now, for the first time, we can credibly claim that if you get cryopreserved you’ll come back not only young and rich, but young and rich and beautiful and thin!

Methinks there must be very few in the Western World today, man woman or child, who can resist a product that has all that to offer – and which, by the way, bestows practical immortality in the bargain.

Ok, Ok, maybe we shouldn’t mention that last part about immortality; it might scare the children.

REFERENCES:

1)     Wiandrowski TP, Marshman G. Subcutaneous fat necrosis of the newborn following hypothermia and complicated by pain and hypercalcaemia. Australas J Dermatol 2001;42:207–10.

2)     Diamantis S, Bastek T, Groben P, Morrell D. Subcutaneous fat necrosis in a newborn following icebag application for treatment of supraventricular tachycardia. J Perinatol 2006;26:518–

3)     Lidagoster MI, Cinelli PB, Levee´ EM, Sian CS. Comparison of autologous fat transfer in fresh, refrigerated, and frozen specimens: an animal model. Ann Plast Surg 2000;44:512–5.

4)      Wolter TP, von Heimburg D, Stoffels I, et al. Cryopreservation of mature human adipocytes: in vitro measurement of viability. Ann Plast Surg 2005;55:408–13.

5)      Manstein D, Laubach H, Watanabe K, Farinelli W, Zurakowski D, Anderson RR. Selective cryolysis: a nivel method of noninvasive fat removal. Lasers Surg Med 2008;40:595–604.

6)     Avram MM, Harry RS. Cryolipolysis for subcutaneous fat layer reduction. Lasers Surg Med. 2009 Dec;41(10):703-8. Review. PubMed PMID: 20014262.

7)     Zelickson B, Egbert BM, Preciado J, Allison J, Springer K, Rhoades RW, Manstein D. Cryolipolysis for noninvasive fat cell destruction: initial results from a pig model. Dermatol Surg. 2009 Oct;35(10):1462-70. Epub 2009 Jul 13. PubMed PMID: 19614940.

8)     Coleman SR, Sachdeva K, Egbert BM, Preciado J, Allison J. Clinical efficacy of noninvasive cryolipolysis and its effects on peripheral nerves. Aesthetic Plast Surg. 2009 ul;33(4):482-8. Epub 2009 Mar 19. PubMed PMID: 19296153.

9)     Fahy GM, Wowk B, Wu J, Phan J, Rasch C, Chang A, Zendejas E. Cryopreservation of organs by vitrification: perspectives and recent advances. Cryobiology. 2004 Apr;48(2):157-78.

Response to Aschwin de Wolf's 'Evidence Based Cryonics'

In his article entitled ‘Evidence Based Cryonics’ Aschwin de Wolf unassailably argues that: “There is an urgent need to move from extrapolation based cryonics to evidence based cryonics. This will require a comprehensive research program aimed at creating realistic cryonics research models. It will also require vast improvements in the monitoring and evaluation of cryonics cases. The current debate should no longer be between advocates and opponents of standby and stabilization but about what stabilization procedures should be used by cryonics organizations given our current knowledge”.

Unfortunately, much of the rest of what he has to say is incomplete or lacks the necessary context required to allow for a fair and technically sound evaluation. Perhaps the brevity of the blog format was the reason for these shortcomings? In any event, I would like to comment on these remarks and provide a somewhat different perspective on the complex and important issues discussed in ‘Evidence Based Cryonics.’

The best place to start is to define what evidence based medicine is, and then proceed to attempt to describe what might constitute ‘evidenced based cryonics.’ Webster’s New World Medical Dictionary, 3rd Edition (2008) defines evidence-based medicine as, “the judicious use of the best current evidence in making decisions about the care of the individual patient. Evidence-based medicine (EBM) is mean to integrate clinical expertise with the best available research evidence and patient values. EBM was initially proposed by Dr. David Sackett and colleagues at McMasters University in Ontario, Canada.” Having defined what EBM is, the next question is, what constitutes “the best current evidence?”

The United States uses the U.S. Preventive Services Task Force (USPSTF) system for evaluating evidence about the effectiveness of medical interventions. The USPSTF classifies evidence in terms of reliability for use in decision making as follows:

* Level I: Evidence obtained from at least one properly designed randomized controlled trial.

* Level II-1: Evidence obtained from well-designed controlled trials without randomization.

* Level II-2: Evidence obtained from well-designed cohort or case-control analytic studies, preferably from more than one center or research group.

* Level II-3: Evidence obtained from multiple time series with or without the intervention. Dramatic results in uncontrolled trials might also be regarded as this type of evidence.

* Level III: Opinions of respected authorities, based on clinical experience, descriptive studies, or reports of expert committees.

* While beyond the scope of discussion here, it is worth noting (and referencing) the work of Guyatt, et al., and the GRADE Working Group in further defining what constitutes the quality and strength of scientific evidence; a formidable and controversial task (1- 6).

* To anyone knowledgeable in the areas of medicine applicable to human cryopatient stabilization and transport procedures (i.e., resuscitation/reanimatology, ischemia-reperfusion injury, solid organ preservation, deep hypothermic cardiopulmonary bypass and whole animal asanguineous perfusion) it will immediately be apparent that none of the 5 classes of evidence presented above can be directly applied to cryonics cases. Arguably, Level III evidence, the “opinions of respected authorities, based on clinical experience, descriptive studies, or reports of expert committees” might apply were there any acknowledged ‘respected authorities’ in the sphere of cryonics standby, stabilization or transport patient care. Alas, no such authorities, respected or otherwise, are currently ‘acknowledged’ to exist.

Thus, the first statement Aschwin makes in opening his article, “Cryonics patients can greatly benefit from rapid stabilization after pronouncement of legal death,” which he defines as “procedures that aim to rapidly restore blood circulation and drop the patient’s temperature” is itself unsupported by either conventional medical research or by cryonics research or case reporting using EBM criteria. If the information-theorertic criteria, as validated by ultrastructural preservation of the brain (7), or the demonstrated recovery of function of the brain are to be used as the gold standards for determining the efficacy of cryonics stabilization and transport procedures, then there currently exists no EBM quality (scientifically robust) data to support “restoration of blood circulation” following pronouncement of medico-legal death in cryopatients.

More specifically, assuming such an intervention is warranted, the question then becomes,’ under what circumstances and in which patients should it be applied?’ Is the patient with 30 minutes of post-arrest warm ischemia better off with simple external cooling followed by cryoprotective perfusion, as opposed to undergoing in-the-field reperfusion using closed chest cardiopulmonary support? What about the patient with profound peri-arrest hypoperfusion with evidence of failed or inadequate brain perfusion, such as the presence of fixed and unresponsive pupils for many minutes, or even for an hour or more, before cardiac arrest occurs and medico-legal death can be pronounced? At what point in the complex and difficult to quantify spectrum of warm ischemic injury should cardiopulmonary support be withheld? Or, given that the benefits of rapid post arrest cooling are unequivocally supported by Level II-2 and Level II-3 evidence from conventional medicine, should such support be modified to mitigate or prevent oxygen-driven reperfusion injury by carrying out CPS under anoxic conditions, and if so, under what circumstances and by what procedures? We have no rigorous answers to these questions and Aschwin is certainly on-point in calling for well designed, cryonics-appropriate studies to answer these and myriad other questions of great importance.

The problem is, as it has been since the inception of clinical cryonics in 1967, “what, if anything, do we do in the meantime?” Indeed, forty-two years later, we have little direct evidence even that cryoprotective perfusion results in superior conservation of identity-critical information under the real-world conditions encountered by today’s cryopatients than would be the case were they subjected to more timely straight freezing!

Is a patient who has suffered hours of warm ischemia better off simply being rapidly cooled and rendered into the solid state, as opposed to being subjected to 24, 48 or 72 hours of cold ischemia, followed by cryoprotective perfusion and freezing or vitrification? How do we even determine what the ultrastructural condition of a brain is following straight freezing? Freezing in the absence of fairly large amounts of colligative cryoprotectant agent(s) results in the collapse of tissue ultrastructure into dense channels of material, the structural condition of which it is currently not possible to determine by techniques such as transmission electron microscopy. Reaching conclusions based on the post-thaw ultrastructure (or lack thereof) of straight frozen tissue is complicated by the potentially myriad artifacts introduced during rewarming, thawing, fixation and embedding required to image tissue ultrastructure.

Given the extreme resource constraints that have historically been present in cryonics, and the lack of directly applicable mainstream medical research, the answer to the question of ‘what to do’ has been to apply reasoned extrapolation of high quality, peer-reviewed biomedical research to the care of the individual cryonics patient, and where possible, to conduct on-point in-house research to validate such armchair speculation.

It is important to point out that since the inception of cryonics in 1964, until approximately 1976, efforts to establish patient care protocols were a group effort between the then extant cryonics societies. The first of these efforts was organized by Robert Ettinger in 1966 and resulted in the protocol developed by Dante Brunol (8). Beginning in 1972, Fred and Linda Chamberlain, Art Quaife, Greg Fahy, Peter Gouras, M.D., Robert Ettinger, and I engaged in an extensive and largely public effort to reach a consensus about what should constitute a good standard of care for cryonics patients based upon extrapolation (and where feasible) experimental validation of findings in the peer-reviewed biomedical literature. This was done via extensive private correspondence, via publication of findings and recommendations in Manrise Technical Review and The Immortalist, as well as in the form of a detailed procedure manual for administering human cryopreservation entitled Instructions for the Induction of Solid State Hypothermia in Humans published by Fred and Linda Chamberlain and available, in part, on-line at: http://www.lifepact.com/mm/mrm001.htm (Readers interested in obtaining a copy of the full manual, for private use, may contact the author at m2darwin@aol.com).

During the 1980s this effort continued and was both documented and subjected to review by the American Cryonics Society, Trans Time, Alcor and Cryovita Laboratories in the form of detailed technical presentations made at the annual Lake Tahoe Life Extension Conferences hosted by Fred and Linda Chamberlain’s Lake Tahoe Life Extension Festivals from 1979 to 1985[1.] An example of such disclosures is available at: http://www.lifepact.com/tahoe.htm.

In short, these efforts were public, largely collegial, and consisted of a best effort to apply insights from the scientific literature to human cryopatients. Furthermore, both Jerry Leaf and I made a sustained and detailed effort to document, by both presentations and publications, the outcomes achieved in detailed human cryopatient case reports (10-20) and in animal studies of post-cryopreservation ultrastructure, including those designed to reproduce conditions encountered under real-world conditions (21, 22).

These efforts resulted in a number of cryopatient stabilization protocols that incorporated multiple drugs to address the multiple mechanisms of ischemia-reperfusion injury as identified in the literature; an approach which Aschwin describes as administration of “an unorthodox number of medications to protect the brain and prevent impairment of circulation. While there are peer reviewed papers that combine a number of medications, there is no precedent in mainstream medicine or biomedical research in using such a large number of medications (in contemporary cryonics, medications protocol exceeds 12 different drugs and fluids).” This statement deserves further scrutiny.

Are poly-drug approaches to treatment unprecedented in medicine? As an example, let’s consider the case of a hyperkalemic hemodialysis patient who experiences cardiac arrest while preparations are being made for emergency hemodialysis. How many and what kind of medications will this patient likely receive in the setting of refractory cardiac arrest? Per the American Heart Association (AHA) Guidelines the patient will initially receive 1 mg epinephrine IV every 3 to 5 minutes during CPR. This may be substituted (after the first dose) with 40 IU of vasopressin IV. Since the patient is in aystole 1mg atropine IV is also given. Concurrent with the administration of these drugs the patient is given 30,000 IU of sodium heparin to allow for the institution of hemodialysis to definitively reduce the serum potassium level. The patient is given an unsuccessful 360 Joule shock at this point. Point of care evaluation of blood electrolytes discloses blood potassium of 12 mmol/L: a level that is incompatible with the return of spontaneous circulation. A decision is made to administer calcium chloride: 5 mL of 10% solution IV over 2 min and 2 amps (60 mlL) of 50% dextrose in water along with 10 IU regular insulin IV (glucose and insulin facilitate a transient profound cellular uptake of potassium from the interstitial and intravascular spaces). CPR is continued for 8 cycles and the patient is again defibrillated with a resulting non-perfusing rhythm consistent with hyperkalemic cardioplegia. CPR is continued while hemodialysis proceeds. After 4 minutes of hemodialysis a third defibrillation attempt is made with the result being coarse ventricular fibrillation. Following another unsuccessful defibrillation attempt, and confirmation by point of care testing that serum potassium has decreased to 7.8 mmol/L with blood pH at 6.95, 300 mg of amiodarone is given in addition to 1 mEq/kg sodium bicarbonate: by slow IV push; the latter to correct the acidosis that has resulted from prolonged CPR and dialysis with a low pH bicarbonate-acetate dialysate.. Following 5 additional cycles of CPR the patient is successfully defibrillated and recovers with a mild neurological deficit as a consequence of extended, low flow perfusion during CPR.

This patient, undergoing routine resuscitation from hyperkalemia cardiac arrest, has just received 9 discrete drugs, all of them indicated, and all of them within the current guidelines for the treatment of hyperkalemic cardiac arrest (23-24). Interestingly, none of these drugs was administered to ameliorate vital organ ischemia-reperfusion injury. The reason for this is that no such drugs are currently clinically available for this indication.

Similarly, patients undergoing acute fluid resuscitation and initial; treatment for septic shock may receive a dozen or more drugs including pressors, ionotropes, a vasodilator, 2-3 antibiotics, insulin, rAPC, and any ancillary drugs required to facilitate renal replacement therapy or mechanical ventilation (see: http://www.leedspicu.org/Documents/Septic%20shock.pdf). So, it is clearly not the case that, “there is no precedent in mainstream medicine” for a multimodal drug treatment approach to complex illness, since multi-drug interventions constitute the standard of care for resuscitation from both cardiac arrest and septic shock and increasingly serve as the backbone of a wide range of successful cancer chemotherapies.

However, it is the case that, at least until recently, multi-drug interventions in biomedical research to treat cerebral ischemia-reperfusion injury have been virtually nonexistent. This is beginning to change as there is increasing understanding of the complex, multifactorial nature of cerebral ischemia-reperfusion injury. Examples of this are the recent successful work of Buckberg, et al in recovering piglets from 90 minutes of deep hypothermic circulatory arrest using a protocol that employed 5 primary therapeutic drugs (plus leukodepletion using a Leukoguard filter in the arterial line during cardiopulmonary bypass) (25), the work of Liu, et al., demonstrating the effectiveness of a combination of cerebral blood flow promoting drugs and the administration of phenyl-N-tert-butyl-nitrone (a free radical inhibitor) and cyclosporine-A (a mitochondrial poration inhibitor) in improving 24 hour neurological outcome after 8 min of experimental normothermic cardiac arrest in pigs (26) and the work of Gupta, et al., combining melatonin and poly (ADP-ribose) polymerase inhibitors in a rat model of stroke – a study that employed 6 drugs in the most successfully treated group (27).Other research combining multiple drugs and other interventions, such as mild therapeutic hypothermia, have also shown positive results (28, 29).

Aschwin goes on to state, “The only existing justification for using current protocol reflects work done at Critical Care Research (CCR) in the 1990s. Although scattered reports exist about the effectiveness of this protocol in resuscitating dogs from up to 17 minutes of normothermic global ischemia, no detailed (peer reviewed) paper has been published about these experiments “

I do not know what is meant by the term “scattered reports” to describe disclosure of this work and would note that there have been two formal public disclosures, the first in the form of United States Patent 5700828 issued on 12/23/1997, and the second in the form of a public seminar which was subsequently distributed as a videotape: Darwin M, Harris, SB, Russell, SR, O’Farrell, Rasch, C, J, Pengelle, C, Fletcher, M. Routine Resuscitation of Dogs from 15-17 Minutes of Normothermic Ischemia (37.5°C) With Long Term Survival (>6 weeks). In: 21st Century Medicine Seminar on Recent Breakthroughs in Cryobiology and Resuscitation Research, Ontario, CA; 1998.

As the principal investigator on this study, I would be the first to agree that it is both regrettable and unacceptable that it has not been either peer reviewed or published. However, as I do not have access to either the primary or the reduced data from this study, I am personally powerless to remedy this situation. Further, I think it extremely unlikely either that I will be given access to this data, or that the results of this study will be published in any meaningful time frame, if at all, by those at CCR who control the study data.

The question thus arises as to whether the drugs identified in this study are of use, either singly or in combination, in the stabilization of cryonics patients? The only certain way to answer that question is to apply them in well designed animal models that closely approximate the spectrum of real-world conditions under which cryopatients eligible for cardiopulmonary support and pharmacological treatment of ischemia-reperfusion injury present for care. Such studies will take tens of thousands of dollars and several years to complete. So, again, the questions arise, ‘what do we do in the meantime ‘and ‘how do we judge the evidence that we use to justify any interventions we undertake?’

It is not possible to answer these questions without considering the specifics of the work in question. Aschwin states that, “in contemporary cryonics, medications protocol exceeds 12 different drugs and fluids” with the implication that the CCR canine resuscitation series (CRS) research was the source of these 12 drugs/fluids, presumably those described by Aschwin in his January 2007 article Human Cryopreservation Stabilization Medications (http://www.alcor.org/Library/html/stabilizationmeds.html).

In fact, the original CRS protocol included a total of 22 drugs!

o Hemodiluent: defined electrolyte-dextran-40 containing solution
o Hypertensives: 3 primary drugs, 1 secondary drug
o Buffer: tromethamine (THAM), 1 drug
o Antiglycemic: 1 drug
o Free radical inhibitors: 6 drugs
o Excitotoxicity Inhibitors: 3 drugs
o Ca++ Antagonists: 1 drug
o Bradykinin Inhibitor: 1 drug
o Leukotriene Antagonists/Inhibitors: 2 drugs
o COX I&II Inhibitors: 1 drug
o Phospholipase Inhibitor: 1 drug
o Antiplatelet: 1 drug
o PARS Inhibitor: 1 drug
o Metabolic Support: 2 drugs

TOTAL: 22 drugs

Of these, 6 drugs (not including the anticoagulant heparin, the hyperosmotic agent mannitol, the flow promoting agent dextran-40, and the buffer THAM, all of which were previously in use in cryonics) were retained in the protocol licensed by CCR to Alcor and to Suspended Animation, Inc. These drugs are s-methylthiourea (SMT), d-alpha tocopherol (Vitamin E), melatonin, alpha Phenyl t-Butyl Nitrone (PBN), kynurenine, and carprofen. How should the utility of these drugs be judged? The first step in such a process is to determine which patients might benefit based on the available information. By definition, only patients eligible for CPS can be treated, since effective use of all of these drugs requires thorough systemic distribution. Patients with 20 minutes or less of normothermic cardiac arrest are probably the only suitable candidates based on the limited ability of closed chest CPS to generate adequate pressure and flow over increasingly long intervals of cardiac arrest. Beyond this general criterion, it is necessary to consider the evidence for the utility of each drug individually, on the basis not only of the CCR study, but in the context of the published literature.

The patent which first discloses the core drugs used in the CCR protocol was United States Patent 5700828 which was filed on 12/07/1995. This is significant because two of the primary cerebroprotective drugs described in this patent, melatonin and PBN, had not been previously demonstrated to be neuroprotective in cerebral ischemia-reperfusion injury. It was not until 2003 that the first peer-reviewed paper documenting the effectiveness of melatonin in ischemia-reperfusion appeared (30) and not until 1999 that the effectiveness of PBN in cerebral ischemia was documented in the literature (31). Since these papers first appeared a vast literature supporting the effectiveness of melatonin in both focal and global cerebral ischemia-reperfusion injury has appeared, and the PBN analog NXY-059 was demonstrated as effective in a wide range of animal models of cerebral ischemic injury (32)., although the drug failed in a RCT of stroke (33).

The utility of vitamin E, mannitol and of dextran-40 in cerebral ischemia reperfusion injury predate the 1995 patent and are extensively documented in the cerebral resuscitation literature. There are few papers documenting the effectiveness of kynurenine, and no papers supporting the effectiveness of carprofen in cerebral ischemia-reperfusion injury, although there are many papers documenting the utility of other non-steroidal anti-inflammatory and NF-kappa B inhibiting drugs in cerebral resuscitation.

Should any or all of these drugs be applied to cryopatients? Aschwin raises a number of possible contraindications which merit consideration: “The lack of relevant published data to support the administration of large numbers of drugs…in cryonics is not just a matter of risking performing redundant procedures. A lot of time and resources are being spent in cryonics on obtaining and maintaining equipment and supplies for these procedures, in addition to the licensing fees paid to use some of these technologies and the training and recruiting of people to perform them. But perhaps the most troublesome problem is that the preparation and execution of these procedures during actual cryonics cases can seriously interfere with rapid and effective cardiopulmonary support and induction of hypothermia.”

It is clear from the foregoing that Aschwin considers immediate post arrest cooling in the presence of CPS to be an essential element of effective cryopatient stabilization. Unfortunately, the use of CPS in this setting carries with it the risks of return of consciousness (33) as well as the return of ‘signs of life’ such as agonal gasping (34, 35), spontaneous movement (36, 37) and even the return of spontaneous circulation. (38). This implies that the cryopatient undergoing CPS must be protected against these undesirable effects by pharmacological intervention. At a minimum, this means that intravenous (IV) or intraosseous (IO) vascular access must be established and at least 3 drugs must be administered (e.g., an anesthetic, a paralytic, and a cardioplegic). Thus, much of the skill, equipment and added personnel required to administer cerebroprotective drugs to cryopatients are, in fact, a requirement of delivering CPS assisted cooling. When Aschwin writes: “the preparation and execution of these procedures during actual cryonics cases can seriously interfere with rapid and effective cardiopulmonary support and induction of hypothermia” it is not clear what he means? Is it establishing IV or IO access, or the administration of a large number of drugs, or both that constitutes a threat to rapid post-arrest CPS and cooling?

CPS and vascular access must, necessarily, proceed together, with CPS (properly) trumping vascular access where any conflict occurs. It should also be noted that CPS, given in the absence of an effective pressor, and (in most cryopatients) volume expansion, will not achieve perfusion that is effective; either for supplying adequate cerebral blood flow to prevent ongoing ischemic injury, or to facilitate heat exchange. CPS implies not only vascular access and the attendant skills, complexity and hardware, but also the administration of at least half a dozen drugs in order to render it both safe and effective. Given this requirement, what are the additional burdens and costs of delivering cerebroprotective medication?

Currently, melatonin, PBN, vitamin E, and carprofen are combined into a single parenteral product by CCR (Vital-Oxy) which can be administered IV or IO via a stopcock manifold using a pressure infuser. Heparin (anticoagulant), vercuronium (paralytic), magnesium sulfate (cardioplegic) and the first dose of vasopressin (pressor) can similarly be combined to create a single parenteral product shortly before use and may be administered ‘push’ via the stopcock manifold. Dexrtran-40 and mannitol may also be combined into a single parenteral product with a total volume of ~550 mL which can also be given via pressure infuser and the stopcock manifold. Bolus, or continuous doses of vasopressin and THAM (buffer), can be given via the same stopcock manifold using battery operated infusion or syringe pumps.

The broader issue to be addressed is how these multiple medications may be given rapidly, accurately, and with the least use of personnel. Compact, battery operated infusion pumps for in-field use are now available, but they cost a fortune. The same is true of programmable, battery operated syringe pumps. I think the solution to this problem is to computerize it. A laptop computer should already be in use during cryopatient stabilization and transport to acquire data from the patient and it can and should be used to give the meds as well. One simple system for doing this would be to use pressure infusers, and syringes under pressure, with open/close line-clamp solenoids under computer control. Meds would be dispensed by the interval of solenoid opening; push meds would be a full open solenoid, and interval bolus meds would be given by briefly, and for a fixed time, opening the solenoid(s). This is an extremely simple system to implement from both the hardware and software standpoints. A schematic of this type of system is shown below:

darwin_meds

Of course, this presumes that the multidrug approach to cerebroprotection of the cryopatient is economically justified. I would be the first to agree that it is not necessary to pay costly licensing fees to derive most of the benefit from the CRS protocol. It is clear from reviewing the literature that the most widely validated and likely most potent drugs in this protocol (in the context of preventing ultrastructural injury secondary to ischemia-reperfusion injury) are melatonin, PBN[2], and, arguably, vitamin E. These are readily available molecules and may be used by any cryonics organization, absent licensing, on the basis of their documented protective effects in the literature. Other likely useful drugs such as dextran-40, THAM and mannitol have a long history of use in cryonics which predates the CCR research and these drugs may also be used at little cost (Darwin M. Transport Protocol for Cryonic Suspension of Humans, Fourth Edition. 1990, http://www.alcor.org/Library/html/1990manual.html).

In the nearly decade and a half that have elapsed since the CCR canine resuscitation series was undertaken many other promising experimental drugs for the inhibition or moderation of cerebral ischemia-reperfusion injury have emerged. I am in complete agreement with Aschwin that the best way to evaluate the potential utility of these drugs to cryopatients is in animal models that are truly relevant and which simulate the actual condition of cryonics patients who present for stabilization and transport. Such patients are typically suffering from extensive activation of the immune-inflammatory cascade, are often severely dehydrated or fluid overloaded, and invariably suffer from serious disturbances in cerebral microcirculation which begin hours or even days before medico-legal death is pronounced. As a consequence, these patients will typically have pre-arrest ischemic injury which will likely be compounded by post-arrest reperfusion. Evaluation of pharmacological interventions should, and indeed properly must be, carried out in animals models that reflect these facts.

Finally, Aschwin writes: “Even more complexity is introduced when cryonics organizations make an attempt to wash out the blood and substitute it with a universal organ preservation solution. The rationale for this procedure is found in conventional organ preservation and emergency medicine research. The question in organ preservation research is no longer whether hypothermic organs benefit from blood substitution with a synthetic solution, but what the ideal composition of such a solution should be. In emergency medicine research asanguineous hypothermic circulatory arrest is increasingly being investigated to stabilize trauma victims. But it is a major step from these developments to the practice of remote blood washout of ischemic patients with expected transport times of 24 hours or more. At present the only sure benefit of remote blood washout is that it enables more rapid cooling of the patient, a benefit that should not be underestimated. But when liquid ventilation becomes available to cryonics patients, rapid cooling rates will be possible without extracorporeal circulation.”

There can be no argument that blood washout followed by long delays to cryoprotective perfusion is deleterious (as currently practiced) on the basis of both clinical experience with cryopatients and recent unpublished animal research by Fahy, et al., of 21st Century Medicine (39). This practice should probably be abandoned until such time as effective solutions are developed for use in cryopatient transports. The statement that “when liquid ventilation becomes available to cryonics patients, rapid cooling rates will be possible without extracorporeal circulation,” is by no means assured. As the primary inventor of fractional tidal liquid assisted pulmonary cooling (40), I feel it is critical to point out that this technique has been validated only in the setting of healthy animals with spontaneous circulation. The reduced flow state attending external CPS and the typically severely injured lungs of the cryopatient present the twin challenges of greatly reduced blood flow coupled with greatly reduced pulmonary surface area (as a consequence of pre-existing or emergent lung injury; i.e., acute respiratory distress syndrome or acute lung injury resulting from closed chest CPS). which will dramatically reduce the efficacy of heat exchange achievable with this technique.

Once again, as Aschwin correctly notes in the context of pharmacological intervention, it is imperative that modalities developed for application in conventional clinical medicine be validated in the very different setting of the patient presenting for cryopreservation after succumbing to prolonged terminal illness – as well as the added insults of peri- and post-arrest systemic and cerebral ischemia.

REFERENCES:

1. Guyatt GH, Oxman AD, Vist G, Kunz R, Falck-Ytter Y, Alonso-Coello P, Schünemann HJ, for the GRADE Working Group. Rating quality of evidence and strength of recommendations GRADE: an emerging consensus on rating quality of evidence and strength of recommendations. BMJ 2008;336:924-926 or [pdf]
2. Guyatt GH, Oxman AD, Kunz R, Vist GE, Falck-Ytter Y, Schünemann HJ; GRADE Working Group. Rating quality of evidence and strength of recommendations: What is “quality of evidence” and why is it important to clinicians? BMJ. 2008 May 3;336(7651):995-8
3. Schünemann HJ, Oxman AD, Brozek J, Glasziou P, Jaeschke R, Vist GE, Williams JW Jr, Kunz R, Craig J, Montori VM, Bossuyt P, Guyatt GH; GRADE Working Group. Grading quality of evidence and strength of recommendations for diagnostic tests and strategies. BMJ. 2008 May 17;336(7653):1106-10
4. Guyatt GH, Oxman AD, Kunz R, Jaeschke R, Helfand M, Liberati A, Vist GE, Schünemann HJ; GRADE working group. Rating quality of evidence and strength of recommendations: Incorporating considerations of resources use into grading recommendations. BMJ. 2008 May 24;336(7654):1170-3
5. Guyatt GH, Oxman AD, Kunz R, Falck-Ytter Y, Vist GE, Liberati A, Schünemann HJ; GRADE Working Group. Rating quality of evidence and strength of recommendations: Going from evidence to recommendations. BMJ. 2008 May 10;336(7652):1049-51
6. Jaeschke R, Guyatt GH, Dellinger P, Schünemann H, Levy MM, Kunz R, Norris S, Bion J; GRADE working group. Use of GRADE grid to reach decisions on clinical practice guidelines when consensus is elusive.
BMJ. 2008 Jul 31;337:a744.
7. Merkle RC: The technical feasibility of cryonics. Med Hypotheses. 1992, 39:6-16.
8. Robert F. Nelson & Sandra Stanley: We Froze The First Man, Dell Publishing Company, New York, 1968, pp. 136-56.
9. Leaf, JD, Cryonic suspension of Sam Berkowitz technical report. Long Life Magazine, 3:(2), March/April, 1979, pp. 30-35.
10. Leaf, JD, Case study: K.V.M. suspension, Cryonics, August 1981, pp. 8-18.
11. Leaf, JD, Quaife A, Case study in neurosuspension. Cryonics. 16 November, 1981 pp. 21-28.
12. Leaf, JD, Darwin, M, Hixon, H, Case report: two consecutive suspensions, a comparative study in experimental suspended animation. Cryonics, August 1981, pp. 8-18.
13. Darwin, MG, Leaf, JD, Hixon, HL, Cryonic suspension case report: A-1133, 08 June, 1987, http://www.alcor.org/Library/pdfs/AlcorCaseA1133.pdf.
14. Darwin, MG, Cryonic suspension case report: A-1108, 08May, 1988, unpublished technical case report of the Alcor Life Extension Foundation
15. Darwin, MG, Cryonic suspension case report: A-1165, 08 October, 1988, unpublished technical case report of the Alcor Life Extension Foundation
16. Darwin, MG, Cryonic suspension case report: A-1169, 21 March 1989, unpublished technical case report of the Alcor Life Extension Foundation
17. Darwin, MG, Cryopreservation patient case report: Arlene Francis Fried, A-1049, 06/09/1990, http://www.alcor.org/Library/html/fried.html.
18. Darwin, MG, Cryopreservation case report: Jerome Butler White, 02-05-1994, unpublished, available from the author upon request.
19. Darwin, MG, Cryopreservation case report: Richard Putnam Marsh, 05-06-1994, unpublished, available from the author upon request.
20. Darwin, MG Cryopreservation of James Gallagher CryoCare patient #C-2150, CryoCare Report Number 6, January 1996, and CryoCare Report Number 9, October 1996, http://www.alcor.org/Library/html/casereportC2150.html.
21. Federowicz (Darwin), MG and Leaf JD, Cryoprotective perfusion and freezing of the Ischemic and nonischemic cat. Cryonics, issue 30, p.14, 1983.
22. Federowicz (Darwin), MG and Leaf JD,. The Effects of Cryopreservation on the Cat. Research reported on Cryonet, December 1992.
23. Part 7.2: Management of Cardiac Arrest, Circulation 2005;112;IV-58-IV-66; originally published online Nov 28, 2005, http://circ.ahajournals.org/cgi/content/full/112/24_suppl/IV-58.
24. Alfonzo, AVM, Simpson, K, Deighan, C, Campbell, S, Fox, J. Modifications to advanced life support in renal failure. RESUS-3067; No. of Pages 17, in press.
25. Buckberg, GJ, Deep hypothermic circulatory arrest and global reperfusion injury: Avoidance by making a pump prime reperfusate—A new concept. J Thoracic and Cardiovasc Surg. 2003;125(3); 625-632.
26. Gupta,S, Kaul, CL, Sharma, S. Neuroprotective effect of combination of poly (ADP-ribose) polymerase inhibitor and antioxidant in middle cerebral artery occlusion induced focal ischemia in rats Neurological Research,26;2004:103-107.
27. LIiu, XL, Nozaria, A, Basu, S, Ronquist, G, Rubertsson, S, Wiklund, L. Neurological outcome after experimental cardiopulmonary resuscitation: a result of delayed and potentially treatable neuronal injury? Acta anaesthesiologica scandinavica,46,(5) 2002:.537-546.
28. Schmid-Elsaesser R, Hungerhuber E, Zausinger S, Baethmann A, Reulen HJ. Combination drug therapy and mild hypothermia: A promising treatment strategy for reversible, focal cerebral ischemia. Stroke 1999, 30: 1891–1899
29. Spinnewyn B, Cornet S, Auguet M, Chabrier PE. Synergistic protective effects of antioxidant and nitric oxide synthase inhibitors in transient focal ischemia. J Cereb Blood Flow Metab 1999; 19:139–14.
30. Reiter RJ, Tan DX. Melatonin: a novel protective agent against oxidative injury of the ischemic/reperfused heart. Cardiovasc Res. 58:10–19, 2003.
31. Shuaib A, Lees KR, Lyden P, Grotta J, Davalos A, Davis SM, Diener HC, Ashwood T,Wasiewski WW, Emeribe U; NXY-059 for the treatment of acute ischemic stroke, N Engl J Med. 2007 Aug 9;357(6):562-71.
32. Siesjö BK, Elmer E, Janelidze S, Keep M, Kristian T, Ouyang,YB et al. Role and mechanisms of secondary mitochondrial failure. Acta Neurochir (Suppl) 1999: 73: 7–13.
33. Darwin, MG, Leaf, JD, Hixon, H, Neuropreservation of Alcor Patient A-1068. http://www.alcor.org/Library/html/casereport8504.html#part2
34. Clark J, Larsen, MP, Culley, LL, Graves, JR, Eisenberg, MS. Incidence of agonal respirations in sudden cardiac arrest. Ann Emerg Med 1992;21(12):1464-7.
35. Rea TD. Agonal respirations during cardiac arrest. Curr Opin Crit Care 2005;11(3):188-91.
36. Jain S, DeGeorgia, M. Brain death-associated reflexes and automatisms. Neurocrit Care 2005;3(2):122-6.
37. Maurino, SJ, Saizar, R,.Bueri.J, Frequency of spinal reflex movements in brain-dead patients. The American Journal of Medicine, 2004, 118(3):311-314;36.
38. Vukmir R, Bircher, N, Radovsky, A, Safar, P. Sodium bicarbonate may improve outcome in dogs with brief or prolonged cardiac arrest. Crit Care Med 1995;23:515-22.
39. Fahy, GM The Whole-Body Vitrification Project at 21st Century Medicine. In the Suspended Animation conference held in Fort Lauderdale, Florida, from May 18th through May 20th May 2007.
40. Federowicz (Darwin), MG, Russell, SR, Harris, SB, Mixed-mode liquid ventilation gas and heat exchange. United States Patent 6,694,977, published 24 February, 2004, http://www.freepatentsonline.com/6694977.html.

ENDNOTES:

[1] The Cryonics Institute declined repeated invitations to participate in these colloquiums.
[2] It is important to note that the PBN used in the CCR study was prepared by dissolving it in boiling water with concurrent microwave heating in the presence of atmospheric oxygen. This is very likely significant because such handling would inevitably create breakdown products of PBN, such as NtBHA and its oxidation product the spin-trap MNP. As Proctor, et al., have pointed out, (Peter H. Proctor and Lynsey P. Tamborello, SAINT-I Worked, But the Neuroprotectant Is Not NXY-059, Stroke 2007 38:e109; published online before print August 23 2007.) it is possible that the failure of NXY-059 in the SAINT-II trial was due to the fact that material used in this trial differed from that used in the successful SAINT-I trial in that it was stabilized and protected against oxidation. It may well be that it is not PBN, per se, that is cerebroprotective, but rather its oxidation and/or break-down products.

Cryonics sets example for emergency medicine

One of the most neglected aspects of cryonics is that its procedures, and the research to support them, can have important practical applications in mainstream fields such as organ preservation and emergency medicine. Contrary to popular opinion, cryonics does not just involve an optimistic extrapolation of existing science but can set the standard for these disciplines. As a matter of fact, that is exactly what cryonics, and cryonics associated research, has been doing over the last 25 years.

The most striking example is the progress in vitrification as an alternative for conventional cryopreservation. Although the idea of eliminating ice formation at low subzero temperatures has been discussed since the beginning of cryobiology, vitrification as a serious research agenda was largely driven by the demand for ice-free preservation of the human brain. Over the last decades this research has culminated in the development of the least toxic vitrification agent to date, 21st Century Medicine’s M22.

The contributions of cryonics to mainstream science and medical practice are not confined to cryobiology. Researchers Jerry Leaf and Mike Darwin made impressive progress in the formulation of bloodless whole body organ preservation solutions to resuscitate dogs from ultraprofound hypothermic temperatures, an intervention that is increasingly being recognized as essential to stabilize trauma victims. In the mid 1990s, Mike Darwin and Steve Harris conceived and developed the idea of using liquid breathing with perfluorocarbons as a method to induce rapid hypothermia. They further validated a multi-modal medications protocol to resuscitate dogs from up to 17 minutes of normothermic cardiac arrest without neurological damage.

Although progress has slowed considerably in the non-cryobiology research areas over the last 10 years, it is encouraging to observe that some of the procedures that are routine in cryonics  stabilization protocol  are starting to catch on in mainstream emergency medicine practice as well. For example, contemporary cryonics stabilization protocol has been strongly shaped by the idea that the best strategy to limit brain injury after cardiac arrest is to combine a number of different interventions: cardiopulmonary support, induction of hypothermia, and administration of circulation-supporting and neuroprotective medications.

It is therefore very encouraging to learn that the Wake County EMS group in North Carolina has achieved impressive results in treating out-of-hospital cardiac arrest victims using a protocol that closely follows elements of current cryonics stabilization protocol. Systematic implementation of immediate induction of hypothermia, continuous compression CPR, and the use of an impedance threshold device (ResQPOD) produced an almost 400% improvement in survival and vast improvements in neurological outcome. A PowerPoint presentation about their experience and protocols are available at their website.

Such real world outcomes do not only inspire confidence in the procedures cryonics organizations can use to protect patients from brain damage after cardiac arrest, it should also serve as a wake-up call to relaunch an aggressive research agenda to push the limits of hypothermic and normothermic resuscitation. In absence of this, it will only be a matter of time before cryonics activists can no longer claim that “we did it first.”

HT Mike Darwin

Early total body washout experiments in cryonics

The question of whether cryonics “works” or not is too general and hides the  point that progressive breakthroughs can make the concept more plausible. Human cryopreservation consists of a number of procedures culminating in long term care at cryogenic temperatures. An evidence based approach to cryonics dictates that the limits of procedures that can be reversed by contemporary technologies should be investigated and pushed further. Under ideal conditions viability of the brain of the patient can be maintained until the early stages of cryoprotective perfusion. In other words, cryonics procedures can be reversed with no or minimal brain injury up until the stage where exposure of the brain to higher concentrations of the vitrification agent compromises viability. Although we do not have a good understanding of the extent of ice formation in the brain of  typical patients in which vitrification is attempted, in ideal cases the current limiting factor to reversibility of cryonics procedures is cryoprotectant toxicity.

During its existence as a research program, cryonics researchers have shown great interest in recovering animals from ultra-profound hypothermic temperatures (lower than 5 degrees Celsius). The ability to routinely lower the temperature of mammals to temperatures close to zero degrees Celsius and  recover them without adverse effects to the brain does make the initial stages of cryonics reversible. Although this procedure does not necessarily require that the blood of an animal is removed at the lowest temperatures, theoretical considerations and experimental evidence dictate that  the use of an universal organ preservation solution will improve outcome. In the 1980s, the Alcor Life Extension Foundation engaged in a series of experiments that recovered dogs perfused with a mannitol-based organ preservation solution called MHP after 5-hours of bloodless perfusion at  5 degrees Celsius without adverse neurological outcome after rewarming.

Less known than those record setting experiments are earlier explorations in cryonics into whole body asanguineous hypothermia. The following document by cryonics researcher and Alcor patient Jerry Leaf documents a Trans Time experiment during the early days of total body washout experiments in cryonics. This account was published in the November/December 1977 issue of Long Life Magazine and the scan contains an introduction by Art Quaife and an afterword by Saul Kent.

Jerry Leaf – A Pilot Study in Hypothermia (1977) PDF

D(+)-Lactose and other sugars in organ preservation and cryonics

A PDF file of this document is available with images and structural visualization of various sugars.

D(+) lactose monohydrate is the principal sugar in mammalian milks. The monohydrate part is easiest to explain; it simply means that the lactose molecule has one water molecule attached to it. This is important because some chemicals can have a lot of water molecules attached to them. For instance, you can have magnesium chloride with two attached water molecules (dihydrate) or six attached water molecules (pentahydrate). This becomes very important when you are weighing out a chemical and you need the chemical to be present in the correct amount. You’ll understand how important this is if you consider that someone proposes to sell you a kilo of some very valuable chemical (say 100 times more valuable than gold per milligram). There is going to be a considerable difference in the amount (by weight and usually by volume) of the actual active chemical you get per milligram or gram (weight) depending upon how hydrated it is (how many water molecules it has attached. The molecular weight (molecular mass) of magnesium chloride is 203.30 and the molecular weight (MW) of water is 18.01. Now, if you have 6 water molecules for each magnesium chloride molecule you have a total mass of water of  (18.01 x 6) = 108.06. That means if you have the pentahydrate salt of magnesium chloride you must add the weight of the 6 water molecules to the MW of magnesium chloride: 203.30 + 108.06 = 311.36. So, if someone is selling you a gram of magnesium chloride pentahydrate at the same price you can buy magnesium chloride anhydrous (no water) you are getting cheated because you are paying the same price for a gram of product that is 1/3rd water!

In biology and chemistry the same principle applies because if you need a certain amount of a chemical for critical reasons, say to maintain normal cell function or inhibit cell swelling in hypothermia, then you must account for any water molecules that may be attached to the chemical. In the case of magnesium chloride pentahydrate versus anhydrous magnesium chloride you are going to have to weigh out about 1/3rd more of the powder of the pentahydrate salt in order to get the same amount of magnesium chloride present in one gram of the anhydrous salt.

Why have pentahydrate, monohydrates, dihydrates and so on of chemicals? The answer is that some chemicals are almost impossible to handle in room air without rapidly absorbing water. Some chemicals will absorb just so much water and no more and thus are very stable under conditions of normal use, so they are supplied in this form. Some chemicals, especially organic chemicals, are virtually impossible to economically produce without one or more attached water molecules. Magnesium chloride is a really good example because it is intensely hygroscopic; it will literally pull water out of the air right before your eyes. So, not only is anhydrous magnesium chloride more expensive than the pentahydrate, it is virtually impossible to handle. If you try to weigh it out it will literally be grabbing water from the ambient air so fast that you can’t tare it on the scale. In seconds you will see tiny droplets of water on the weighing boat or paper where the magnesium chloride has literally pulled so much water out of the air it is fully dissolved in a tiny droplet of water! Calcium chloride is just about as bad, so, you’ll notice that we don’t even bother trying to weigh these chemicals out as dry powders, but rather buy them as pharmaceutical products for injection because they are already dissolved in solution in very precise concentrations. Thus, it is much simpler to draw up the correct volume of these salts dissolved in solution to add the desired amount of these two chemicals to perfusate. It is possible to weigh them, but you have to be quick and it helps if you live the desert where the humidity is very low.

Now we come to the D(+) part which is much harder to explain. In the early part of the 19th Century the French physicist Dominique F.J. Arago noticed that when he passed polarized light through quartz crystals the light could be rotated either to the left or right depending upon the individual crystal. Shortly thereafter the brilliant physicist and mathematician Jean Baptise Biot (the Biot number, a dimensionless number used in unsteady-state (or transient) heat transfer calculations, is named after him) also observed this same effect in liquids and gases of organic substances such as turpentine and some other petroleum products. About 10 years later the English astronomer Sir Joun F.W. Herschel discovered that different crystal forms of quartz rotated the linear polarization in different directions. Simple polarimeters have been used since this time to measure the concentrations of monosaccharide sugars, such as glucose, in solution. In fact, one name for glucose, -dextrose-, is so named because it causes linearly polarized light to rotate to the right or “dexter” side. Similarly, levulose, more commonly known as fructose (fruit sugar) causes the plane of polarization to rotate to the left. Fructose is even more strongly levorotatory than glucose is dextrorotatory. Invert sugar which is formed by adding fructose to a solution of glucose, gets its name from the fact that subsequent structural conversion causes the direction of rotation to “invert” from right to left.

The reason for this behaviour of these seemingly identical substances was not understood until the mid-19th Century when Pasteur was working on the problem of why wine was souring as opposed to fermenting into an alcohol solution. The culprit was yeast that metabolized the fructose in the grape juice to tartaric acid. A solution of tartaric acid derived from living things (the wine lees yeasts) rotated the plane of polarization of light passing through it, whereas chemically synthesized tartaric acid prepared by non-organic means in the laboratory did not have this effect. This was puzzling because both the synthetic and the biologically derived tartaric acid undergo the same chemical reactions and are identical in their elemental (atomic) composition. Pasteur noticed that the crystals came in two asymmetric forms that were mirror images of one another. He meticulously sorted the crystals by hand and then dissolved each of the two forms of crystals in water; solutions of one form rotated polarized light clockwise, while the other form rotated light counter-clockwise. An equal mix of the two had no polarizing effect on light. Pasteur deduced the molecule tartaric acid molecule was asymmetric and could exist in two different forms that resemble one another; as would left- and right-hand gloves, and that the organic form of the compound consisted purely of the one type.

This phenomenon is referred to as isomerism and occurs when two molecules have the same molecular formula (atomic composition) yet have different structures and therefore different chemical and physical properties. There are many different kinds of isomers. The two major divisions of isomers are the geometric and the structural. Structural isomers are isomers that have the same number of atoms but different arrangement of atoms. One structural isomer of glucose is fructose. Geometric isomers are identical in arrangement of covalent bonds but are different in the order that the groups are arranged.

A major category is stereoisomers which are two isomers that have the number of atoms in the same order. A stereoisomer of glucose is galactose. In the Fischer projection all of the atoms are the same except for one rotated group. There are two categories of stereoisomers, enantiomers and diastereomers. Enantiomers are two isomers that are mirror images of each other when looked at in 3D while diastereomers are not. Galactose is just one of many diastereomers of glucose. To find out the possible number of stereoisomer forms a monosaccharide can have, you can use the formula 2x where x is the number of chiral carbons the molecule has. Molecular chirality occurs when a sugar has a carbon with four different groups attached to it. Any carbon with a double bond on it is never chiral nor are the end carbons. Because glucose has four chiral carbons there are 24 different stereoisomers; which means that there are sixteen different stereoisomers for glucose.

Two of the main divisions of glucose’s many forms are l-glucose and d-glucose. These two are enantiomers which are determined by whether the two molecules are symmetrical at the last chiral carbon. When the hydroxyl group is on the last chiral carbon on the right it is considered d-glucose  and when it is on the left it is classified as l-glucose. The “d” means that the glucose rotates polarized light to the right (dextrorotatory) and the “l” stands for  levorotary (rotates polarized light to the left). These refer to how a plane of light rotates as it passes through a solution of it. First light is passed through a polarizing filter, then a polarimeter containing a solution made with the molecule. When a d-solution is in the polarimeter it will cause the light to turn to the right or at positive angle, while an l-solution will cause the light to turn to the left or a negative angle. Both d-glucose and l-glucose exist naturally but d-glucose, also called dextrose, is the most abundant sugar on the planet.

The practical biological and chemical implications of these isomeric structural differences is profound. D-glucose (dextrose) is the principal sugar used by the body to generate energy. By contrast, l-glucose cannot be significantly metabolized and an animal or human would starve to death if this was the only carbohydrate available in its diet and no other sources of energy (fats or proteins) were available. L-glucose looks, tastes and has the same mouth feel as d-glucose and there has been considerable interest in producing it in large quantities as an artificial sweetener.  Unfortunately, the synthetic pathway to produce l-glucose, and more importantly, the separation of the d- and l-glucose isomers after synthesis is currently prohibitively expensive.

What does all this have to with cryonics and organ preservation? Under normal metabolic conditions the cells of the body produce chemical energy in the form of ATP and about 1/3rd of this energy is used to pump ions into and out of the cells. This is necessary because the most common salts (ions) are very small and can easily pass through the cell membranes. Two straightforward examples are very much on-point. Cells need high concentrations of the potassium ion inside them to be able to function properly including carrying out some vital chemical reactions and doing things like contracting in the case of muscles or transmitting signals in the case of nerve cells. Conversely, cells must not have too much sodium in them or they  become swollen (edematous) and while this can ultimately rupture or lyse the cell, long before this happens cell swelling disrupts the meshwork of supports that maintain the cell’s shape and probably serve as scaffolding for various enzymes to be anchored on and to facilitate efficient chemical processing (metabolism). Unfortunately, sodium has a net negative charge and the protein inside cells has a net positive charge. Thus, sodium will flow into cells and carry water with it resulting in cellular edema. This process is prevented by active pumping of sodium out of the cell. Similarly, calcium is extremely toxic to cellular mitochondria in high concentrations and calcium is also used as a critical signalling molecule inside cells. Thus, the calcium concentration outside cells is typically 10,000 times higher than that present inside cells. Again, this difference in concentration is maintained largely by active pumping which requires energy expenditure and on-going metabolism.

So, sodium gets pumped out and potassium gets pumped in and this process is linked and carried out by the same molecular machine; the sodium-potassium pump. Of course, all of this presumes that there is both available energy in the form of ATP and that the cellular pumping machinery can use that energy. There are a number of things that can interrupt ion pumping. There can be a lack of energy due to starvation, hypoxia or ischemia, and there can exist situations where the energy is available but cannot be used. Some chemicals poison enzymes critical to ion pumping; a classic example is tetrodotoxin which comes from blowfish and which poisons sodium pumping. The other condition where adequate energy (ATP) can exist but cannot be used is deep hypothermia. Non-hibernating animals have enzymes that shut down or become inactive when the temperature is reduced well below that of normal body temperature. In humans (and most non-hibernating mammals) the  enzyme responsible for pumping sodium out of cells and potassium into them, sodium-potassium-ATPase, is largely inhibited at 10oC and is virtually shut down at few degrees above 0 oC.

Cell swelling in brain cells occurs with incredible rapidity after interruption of blood flow in ischemia (cardiac arrest). While cell swelling is not the only, or even primary, cause of injury in cerebral ischemia, it is a major player in causing injury in cold ischemia; conditions which obviously obtain in organ preservation and ultra-profound hypothermia in cryonics patients. The way this edema is prevented in organ preservation is to replace almost all of the small cell membrane permeable ions with big molecules that cannot pass through the cell membrane and which are osmotically active; in others words can hold water outside of the cell.  The first solution to do this with any success was Collin’s Solution invented by Geoff Collins. It used comparatively large phosphate salts to keep water outside of the cells and prevent cellular edema. However, phosphates do leak across the cell membrane and they are incompatible with DMSO and also precipitate as crystals when solutions are cooled to low temperatures or frozen.

Thus, the organ preservationists turned to sugars and sugar alcohols as molecules to serve as an osmotic agent and prevent cell swelling. Sugars are comparatively large molecules and some are very large. They do not typically pass through cell membranes rapidly, if at all. Some of the first sugars tried were glucose and sucrose and the sugar-alcohol mannitol. Neither glucose nor sucrose worked well. Glucose leaks across cell membranes fairly rapidly and has facilitated transport in the brain. Sucrose makes quite viscous solutions in the necessary concentrations (~180 mM) and for unknown reasons is toxic to the kidney tubule cells. Mannitol was much more successful in the laboratory but never made it into clinical organ preservation solutions.

In the 1980s, a biochemist named Jim Southard and a transplant surgeon named Folkert Belzer began to systematically study molecules to inhibit cold ischemic swelling, as well as other molecules to help conserve ATP, inhibit free radical damage, and otherwise address the derangements that occur under deep hypothermic conditions. They identified two sugars as particularly effective in inhibiting cold ischemic cellular edema, raffinose and lactobionate. They combined these two sugars along with other ingredients to create the first and still most successful “universal” organ preservation solution, UW-Solution, or as it is commercially marketed, Viaspan.

Unfortunately, ViaSpan does not work for the brain. We tried it extensively in the early 1990s and got serious cerebral edema followed by convulsions and death in dogs that had been perfused with ViaSpan for as little as two hours! By contrast, we could recover dogs perfused with MHP (a mannitol based perfusate) after 5-hours of bloodless perfusion with the solution at  5oC with no neurological or other problems; most of the dogs were placed with cryonics members and lived out the rest of their lives normally.

Recently, 21st Century Medicine has been systematically investigating hypothermic organ preservation and they have made a number of stunning breakthroughs. One thing which was long overdue to be done was to systematically screen various molecules for their cell swelling inhibiting effects. They found that one sugar in particular was highly effective, D(+)-lactose. Only the d-isomer worked well.

Why some sugars work and others do not, or actually cause harm, is a mystery. The molecular weight is certainly a factor, but different sugars with nearly identical molecular weights may perform totally differently. Also, the isomer of the sugar appears critical in some cases, as is the situation with lactose.  21st Century Medicine has patented an organ preservation based on D(+)-lactose and is in Phase II clinical trials for this solution, which they call TranSend. They are currently getting 72-hour simple flush and store on ice preservation of kidneys (rabbit and dog), pancreases and livers (dogs) and are getting similar results in the human clinical trials. They have achieved 48-hour heart preservation using a derivative of this solution which combines periods of trickle-flow cold perfusion with brief intervals of modest warming to ~15 oC.

Dogs resuscitated after 3 hours of cardiac arrest from exsanguination

Despite sensational news items about “zombie dogs,” biomedical researchers and clinicians have known for a long time that interruptions in consciousness and blood circulation can be reversed without neurological deficits, provided such events do not produce ischemic injury. There are even species who can enter a state of reversible metabolic arrest such as tardigrades (water bears). Naturally, researchers have recognized the opportunities that depressed metabolism holds for stabilizing trauma victims.  Although the jury is still out on the question if therapeutic hypometabolism can be induced in large animals by chemical means, there are no doubts that  lower temperatures reduce metabolism, allowing patients to tolerate longer periods of circulatory arrest.

The published record for reversible hypothermic circulatory arrest is 3 hours in a canine model. A recent study by Wu, Drabek, Tisherman et al. (2008) documented resuscitation from 3 hours of exsanguination cardiac arrest  (2.5 hours of no flow) after rapid induction of profound hypothermia using cardiopulmonary bypass. These results are quite impressive in light of the fact that in the latter study cardiac arrest was induced at normal body temperature by exsanguination, and the organ preservation solution to replace the blood consisted of just chilled saline plus dissolved oxygen and/or glucose. The authors attribute their success in extending satisfactory neurological recovery from 2 to 3 hours of exsanguination cardiac arrest to the addition of energy substrates, and oxygen in particular, during induction of profound hypothermia.

Research of this nature benefits cryonics in a number of ways. If hypothermic circulatory arrest will become routine in emergency medicine and military medicine, the general public will get increased exposure to the fact that circulatory arrest does not equal death. Research of this nature also demonstrates that induction of ultra-profound hypothermic arrest in humans may be reversible and therefore the initial stages of cryonics stabilization procedures as well. The more practical application is that it offers the prospect of extending the period the brain can be kept viable after pronouncement of legal death during remote transport of cryonics patients. Last but not least, this specific study provides optimism that viability of the brain can be maintained if hypothermia is induced after circulatory arrest, provided metabolic support is given and cooling rates are fast enough to avoid irreversible injury to the brain.

Unfortunately, the technical capability to reverse 3 hours of asanguineous hypothermic arrest falls short of what is needed for cryonics patients who are stabilized in remote locations. Transport times between the location of cardiac arrest and the cryonics facility often exceed 24 hours.  Although loss of viability of the brain does not constitute information-theoretic death, it would be desirable if cryonics organizations would be able to routinely secure viability of the brain between pronouncement of legal death and start of cryoprotectant perfusion.

Such advances will require substantial investments into  the development and implementation of improved organ preservation solutions, perfusion techniques, and resuscitation protocols. Potential directions  for such research include addition of effective neuroprotective compounds and “hibernation mimetics” to the organ preservation solution and low flow or intermittent perfusion during patient transport.

In 2005, when asked to comment on the prospects of using hypothermic circulatory arrest to treat trauma victims, Dr. Thomas Scalea, physician-in-chief at the R. Adams Cowley Shock Trauma Center at the University of Maryland Medical Center, was reported saying:

“As potentially crazy as this might sound, you’re comparing it against essentially certain death, so it’s hard to see how we can do any worse….all of us are incredibly energized by the thought of being able to do better.”

Such reasoning should equally apply to the practice of human cryopreservation, which employs even lower temperatures to protect people against “essentially certain death.”

Induction of hypothermia before CPR improves survival

It is difficult to match concerns about reperfusion injury during cardiopulmonary resuscitation (CPR) with specific proposals for alternative interventions. After all, no matter how harmful the effects of oxygenation may be, not restoring circulation in a patient in cardiac arrest is hardly a credible option. One alternative would be to restore circulation but withhold oxygen (or ventilate with room air). Another alternative would be to induce hypothermia during circulatory arrest before restoring circulation.

A recent paper in Resuscitation investigated the latter option and reports that delaying reperfusion  in mice until induction of mild hypothermia has been achieved can improve hemodynamics, survival and neurological outcome.  The time to drop the temperature from 37 degrees Celsius to 30 degrees Celsius was 90 seconds in mice. As the authors note, “this is not currently feasible in humans and it is likely that much longer resuscitation delays in the clinical setting might counteract the benefit of cooling before ROSC (return of spontaneous circulation)”.

Rapid partial cooling (as the authors suggest) may solve this problem but restoring circulation will result in moving warm blood to the very organs (such as the heart and the brain) that just had been cooled. Such an intervention will only work if some of the protective mechanisms of hypothermia, such as altered gene expression, are (partially) retained during subsequent rewarming.

One treatment modality that the authors did not research, but warrants investigation, would be to “mimic” intra-arrest hypothermia by restoring circulation and giving a cocktail of neuroprotective agents prior to restoring oxygenation. Such an approach may not eliminate all free radical injury upon restoring circulation, or eliminate other elements of reperfusion injury such as calcium overload and inflammatory responses, but it might be an interesting treatment to compare with induction of intra-arrest hypothermia and delayed CPR.

Critical cooling rate to prevent ischemic brain injury

Induction of hypothermia can reduce injury to the brain when it is deprived of oxygen. How fast do we need to cool a patient during cardiac arrest or stroke to prevent irreversible injury to the brain?

It is an established fact that induction of hypothermia prior, during, or after circulatory arrest can reduce brain injury. As a general rule, the lower the temperature is dropped, the longer the brain can tolerate circulatory arrest. The neuroprotective effects of hypothermia are often expressed using the Q10 rule which says that for every 10 degrees Celsius drop in temperature metabolic rate decreases by 50%. Or to put it differently, the Q10 rule states that ischemic damage susceptibility is decreased by a factor of 2 for every 10 degrees Celsius temperature drop.  Q10 may vary between species and in different organs and cells. For example, different temperature sensitivities were observed for release of the neurotransmitters glutamate, aspartate, glycine, and GABA during cerebral ischemia by Nakashima et al. Because even very modest reductions of brain temperature can have profound neuroprotective effects, the Q10 rule may not tell the complete story.

Other things being equal, it would be very useful to have a measure of brain injury when hypothermia is induced prior and/or during cardiac arrest. At least two authors have made an attempt to produce such a measure of ischemic exposure. In Cryonics Magazine (2nd Quarter, 1996), Michael Perry started initial work on this in an article called “Toward a Measure of Ischemic Exposure” (PDF).  Perry’s Measure of Ischemic Exposure (MIX) calculates how long the patient has been at a given temperature, with a higher weighting used for higher temperatures. A related measure has been proposed be Steve Harris called the E-HIT. E-HIT stands for Equivalent Homeothermic Ischemic Time. In his (unpublished) manuscript, Harris uses the E-HIT formula to calculate the equivalent normothermic ischemic time for different cryonics case scenarios and real cases. Clearly, the availability of such a measure (and its routine calculation in case reports) would constitute a major contribution to cryonics as evidence based medicine. It could aid in deciding if viability of the brain was maintained during cryonics procedures by estimating the equivalent warm ischemic time.

What makes such a measure complicated during cardiopulmonary resuscitation (CPR), or cardiopulmonary support (CPS) in cryonics stabilization procedures is that hypothermia may only constitute one intervention to mitigate brain injury. In an ideal cryonics case, pronouncement of legal death is followed by rapid restoration of oxygenated blood flow to the brain by (mechanical) cardiopulmonary support, administration of neuroprotective drugs and induction of hypothermia. Such a combination of interventions might avoid any injury to the brain, reducing the equivalent warm ischemic time to zero. A more realistic scenario is that such a combination of interventions may reduce the extent of ischemic injury compared to cooling only. Another complicating factor is that oxygenation in combination with low perfusion pressures might produce more injury than “anoxic cardiopulmonary support” (chest compressions without ventilation). It is clear that calculating a measure of equivalent ischemic time for real cryonics cases can become very complicated.

It would be interesting to know the cooling rate that would be necessary to stay ahead of brain injury, using contemporary medical criteria, during circulatory arrest. For this purpose we use some very simplifying assumptions:

1.The patient is not ischemic prior to pronouncement of legal death.

2. Cooling is initiated immediately after pronouncement of legal death.

3. There is no cardiopulmonary support or administration of neuroprotective agents.

4. Brain injury starts at 5 minutes of warm ischemia.

5. Q10 is 2.0: for every 10 degrees Celsius we decrease the temperature , metabolism is dropped 50% , which doubles the time a patient can tolerate ischemia.

6. No other forms of injury occur other than ischemic injury.

7. Ischemic injury is completely eliminated at the glass transition temperature of the vitrification agent M22 (-123.3°C).

8. A constant cooling rate is assumed.

Using these assumptions, Alcor’s Mike Perry calculates that a cooling rate of 2.89 degrees Celsius per minute is necessary to stay ahead of the equivalent of 5 minutes of warm ischemia.

Let Ehit = total ischemic time limit in hours, 1/12 corresponding to 5 min
Q10 = factor of decrease in metabolism per 10 degrees
Tdrop = desired temperature drop, from 37 degrees (body temp) down to -123.3= 160.3 degrees Celsius
ch=desired cooling rate in deg/hour
cm=desired cooling rate in deg/min = ch/60

Then

ch = 10*(1-exp(-Tdrop*ln(Q10)/10))/(Ehit*ln(Q10))

For Q10=2, Tdrop = 160.3, cm = 2.89 deg/min

If some of the assumptions are slightly changed we find the following for Q10=2.2

For Q10=2.2, Tdrop = 160.3, cm = 2.54 deg/min

If we assume negligible ischemic insult below 0 Celsius and only worry about cooling down to that temperature, so Tdrop is only 37 rather than 160.3, it doesn’t change these amounts drastically:

For Q10=2, Tdrop = 37, cm = 2.66 deg/min
For Q10=2.2, Tdrop = 37, cm = 2.40 deg/min

Clearly, such high cooling rates cannot be achieved during either conventional cardiopulmonary resuscitation or cardiopulmonary support in cryonics. The cooling rates we can hope for during the initial stages of cryonics procedures may exceed 1.0 degrees Celsius per minute at best. It is therefore not realistic to assume that cooling alone may be able to limit brain injury to a degree that allows resuscitation without adverse neurological effects using contemporary medical criteria. This should strengthen the case for the use of other interventions such as administration of neuroprotective agents and oxygenation of the patient. Although the latter intervention may produce adverse effects on the brain itself, the calculations above indicate that anoxic cardiopulmonary support is not compatible with maintaining viability of the brain as the objective of cryonics stabilization procedures. The case for rapid stabilization of cryonics patients remains strong.

H.P. Lovecraft's "Cool Air" and cryonics

In “Heritage of Horror,” Lovecraft scholar S.T. Joshi writes that Lovecraft’s short story “Cool Air” “anticipates cryogenic research.” We can forgive Joshi the common mistake of writing “cryogenics” when he means “cryonics,” but how much cryonics is there really in Lovecraft’s “Cool Air?”

“Cool Air” (1926) tells the story of a struggling writer who has secured affordable housing in a converted brownstone on West 14th Street in New York City to devote himself to “dreary and unprofitable magazine work.” Around three weeks pass when an incident in the room above introduces the reader to the character of Dr. Muñoz, whose “complication of maladies” requires an environment of constant cold. When the main character experiences a sudden heart attack, his initial repugnace for the eccentric doctor changes to admiration when Dr. Muñoz is able to offer him relief with a suitable combination of drugs.

Dr. Muñoz, we learn, is the “the bitterest of sworn enemies to death, and had sunk his fortune and lost all his friends in a lifetime of bizarre experiment devoted to its bafflement and extirpation.” He believes that “will and consciousness are stronger than organic life itself, so that if a bodily frame be but originally healthy and carefully preserved, it may through a scientific enhancement of these qualities retain a kind of nervous animation despite the most serious impairments, defects, or even absences in the battery of specific organs.” As the story develops we learn about the doctor’s own (increasing) need for a cold environment to preserve his bodily frame.

Just as in cryonics, Dr. Muñoz employs cold to prevent decomposition. And decreased temperatures confer increased benefits in slowing down the rate of decomposition. In cryonics these benefits of low temperatures are exploited by reducing the temperature of the patient to a point of complete metabolic arrest. At the temperature of liquid nitrogen (-196 degrees Celcius) biological time stands still for all practical purposes.

But what is remarkable about Dr. Muñoz’s approach is that he reaps the metabolic advantages of induced hypothermia without these temperatures preventing his mind from functioning. Dr. Muñoz seems to be unusually “alive” at ultra-profound, or even, high subzero temperatures! Because the EEG of a human brain becomes flat below 20 degrees Celcius, some other process must be involved, perhaps the “incantations of the mediaevalists, since he believed these cryptic formulae to contain rare psychological stimuli which might conceivably have singular effects on the substance of a nervous system from which organic pulsations had fled.”

Unless Dr. Muñoz’s treatment induced profound changes in the body’s biochemistry that allowed it to operate at much lower temperatures, his philosophy of life seems less “materialistic” and coherent than that of Lovecraft’s other enemy of death, Herbert West. Lovecraft never anticipated the practice of cryonics in a systematic fashion, but if Dr. Muñoz and Herbert West could have put their brilliant minds together, the benefits of cold temperatures could have been reaped to induce metabolic arrest in anticipation of future resuscitation of the “dead.”