CHAPTER II
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If you are about forty years old now, then probably when you die, in another thirty or forty years, physicians or technicians paid by your insurance company will bank your blood, perfuse your parts, and lay you to rest - not eternal rest, but temporary, and not in the cold ground, but in a much colder freezer. A few years later, perhaps they will slide your wife in beside you.
At first thought, many people find this notion both implausible and a little repellent. They may find it repellent because their minds associate a freezer with dead meat. They find it implausible, because they know a lamb chop looks pretty inert to begin with, and furthermore begins to spoil after a very few years in a freezer at 0 degrees farenheit.
It is also recalled that we sometimes have to chop off a severely frostbitten toe; we cannot revive it, even though the rest of the body is alive. How, then, can we hope to revive a man frozen throughout his very vitals? How can we have any confidence that it will ever be possible?
A mere, generalized optimism is certainly not convincing. It is all very well to say that future science will surpass imagination; but will it be able to take a tub of frozen corned beef hash, and from this reconstitute a steer-the same steer that went into the hash? We are interested in something that is probable, and not just barely conceivable. If our chances were no better than those of the hypothetical steer, we would not want to be bothered.
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To provide a basis for reasonable confidence, let us examine carefully some of the salient facts and estimates concerning the effects on living animals of cooling and freezing.
Our basic argument was based on one fact and one assumption. The fact - that it is possible, right now, to preserve dead people with essentially no deterioration, indefinitely - is easily established.
It is a well-known principle of chemistry that at temperatures near absolute zero (about -273C or -459F) reaction rates generally become negligibly small. The molecules are nearly motionless. The life processes of any organism cooled near this extreme should become immeasurably slow, and also any processes of decay. Actual observation confirms this theoretical principle.
Dr. Harold T. Meryman (Naval Medical Research Institute, National Naval Medical Center, Bethesda, Maryland), a leading authority in the field, says, "Under any circumstances, storage in liquid nitrogen, at - 197C can be considered as essentially indefinite." (68)
Dr. Humberto Fernandez-Moran (University of Chicago), a prominent expert in biophysics, notes that ". . . no detectable metabolic activity has been reported at liquid nitrogen temperatures . . ." He points out, however, that activity involving short-lived molecular fragments called "free radicals" can occur at - 197C and that long-term storage should perhaps be at liquid helium temperatures, namely within a few degrees of absolute zero. The reaction rates at liquid helium temperatures are calculated to be slower than at liquid nitrogen temperatures by a factor of about ten trillion! (30)
Many other investigators have written to the same effect. The
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consensus of the best-informed opinion, based on long observation as well as theory, indicates that a body cooled by liquid nitrogen can be stored without significant changes or deterioration for a period measured at least in years and probably in centuries. A body cooled by liquid helium will keep, for all practical purposes, forever.
Clearly, then, the storage problem is not the main difficulty. Whatever condition the body is in when it reaches the storage temperature, that is the condition in which it will remain for as long as it is necessary to keep it. If it is alive, it will remain alive; if it is somewhat damaged, it will remain somewhat damaged.
The principal hazards pertain to the freezing and thawing processes. Let us next inquire what progress has been made in actually freezing specimens and restoring them to active life.
Successes in Freezing Animals and Tissues
Among smaller and lower organisms, there are many which can survive actual hard freezing at temperatures far below the freezing point, even without any special protection, and others which can be assisted to do so.
Becquerel has found that certain minute, primitive animals, which can tolerate dehydration, can be cooled, after drying, to within a fraction of a degree of absolute zero, and after rewarming and remoistening revive fully. (5) Since the water had been removed before freezing, there had been no damage from ice crystals.
Two Japanese scientists, Asahina and Aoki, worked with larvae of a certain insect, Cnidocampa flavescens. The larvae were removed from their cocoons, kept for one day at -30C, and then immersed in liquid oxygen at - 180C. After thawing, their hearts resumed beating, and some of them lived to their next developmental stage, that of "imago," but none completed meta-
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morphosis to the adult stage. (2) It was thought that the pre- freezing period of one day at -30C allowed growth of ice crystals outside, rather than inside, the cells; that is, the ice crystals formed in the intercellular spaces.
Many protective agents have been tried to reduce damage to animal tissues in freezing; perhaps the most successful of these has been glycerol. The first evidence was provided by Professor Jean Rostand, working with frog spermatozoa; motility of the sperm was preserved for several days at -4C to -6C. (94) (The freezing point of pure water at standard pressure is 0C.) Subsequently it has been found that certain cold-hardy insects naturally contain glycerol in their bodies! (110)
Another protective agent sometimes used successfully is ethylene glycol, a solution of which was used by Dr. B. J. Luyet and Dr. M. C. Hartring in freezing vinegar eels, anguillula aceti. The eels survived immersion in liquid air at about - 190C, provided both cooling and rewarming were rapid. (110) It was thought that the ethylene glycol caused dehydration, and induced a vitreous rather than a crystalline condition of the water in the cells.
Clams on certain northern shores, exposed to temperatures far below 0 degrees celsius when the tide runs out, apparently become solidly frozen and thawed twice daily for weeks on end, yet survive. It is suspected that these organisms also may secrete a natural protective agent of some kind, and investigation is continuing. (110)
When we turn our attention to larger and more highly developed forms of life, we find there have been many successes in freezing and reviving cells, tissues, and even organs. Usually protective agents have been required, but not in all cases.
Bull semen has been treated with glycerol, stored at -79C (the temperature of solid carbon dioxide or "dry ice") for periods up to seven years, and thawed with a high survival rate. But it is interesting to note that a little deterioration occurs even at this
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temperature; lower temperatures improve the results. (110) It is also observed, contrary to the experience with vinegar eels, that too rapid freezing can be harmful. (110)
Human spermatozoa, without protection, show resistance to extreme cold which varies from cell to cell, and also from donor to donor. In one study, up to 10 per cent of the sperm cells survived five-minute exposure; hardihood varied from donor to donor, but for a single donor survival was the same at -79C, -196C, and -269C. (110)
Dramatic evidence of the viability of deep-frozen human sperm is furnished in a New York Times Service article (Detroit Free Press) of September 6, 1963. Two babies were horn to women who had been artificially inseminated with sperm stored for two months at liquid nitrogen temperature. Dr. Jerome K. Sherman, of the University of Arkansas, is said to have stored semen at this temperature for three and a half years without loss.
Dr. S. W. Jacob and co-workers have reported cooling human conjunctival cells (from the membrane lining the eyelid) as well as sperm to within less than one degree of absolute zero, with viability maintained. (50)
Embryo chicken hearts, after treatment with glycerol solution, have been cooled to -190C, and heating resumed after thawing. This was one of the developments which led Dr. D. K. C. MacDonald of Ottawa University, an expert in low-temperature physics, to write, . . . perhaps the day will come when, if you want it, you can arrange to 'hibernate' for a thousand years or so in liquid air, and then be 'wakened up' again to see how the world has changed in the meantime. (65)
In the case of the mammals, attempts to freeze, store, thaw, and revive specimens have not yet been completely successful. But there have been many partial successes, and much has been learned.
The best-known experiments may be those of Dr. Audrey U.
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Smith, of the National Institute for Medical Research, Mill Hill, London, working with golden hamsters. These animals have been successfully revived after being about half frozen. In particular, more than half the water in the brain had changed to ice, and the bodies were rigid; yet these mammals recovered to apparently normal activity. (110) This is very important, since it seems to provide some evidence that mental faculties can survive freezing and thawing.
It is to be noted that Dr. Smith's results were achieved by crude means: the cooling was with cold baths and cold packs, and the aids to resuscitation were simply artificial respiration and microwave diathermy. The tissues were not given any local protection in the form of special infusions, although it is known that such protection can be very important.
Similar work includes that of Andjus and Lovelock, who have reported recovery and long-term survival of 80 per cent to 100 per cent of ice cold rats. (110) Dr. J. R. Kenyon and his co workers have chilled dogs approximately to the freezing point, with heartbeat and circulation completely stopped, and obtained sufficiently complete recovery so that they survived many weeks after the experiment. Chemical infusions were used to counter- act accumulation of certain harmful metabolic products. (55)
The mechanism of freezing damage is still poorly understood. There is much variation in hardihood among different types of cells, and even among individual cells of the same type. Different temperature ranges also have their own distinctive problems.
Experimental work directed toward testing new theories and new protective agents and techniques proceeds vigorously, but on a relatively small scale. When the public becomes interested in freezers, progress should become much swifter. It is not always possible to hasten scientific progress simply by spending more money, but in this instance the possibility seems to exist. Many avenues apparently are not being explored, for lack of workers.
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Among other things, a massive, systematic search for new protective agents seems called for.
Even with work at the present relatively slow pace, there is much optimism. Dr. A. S. Parkes, F.R.S., in the foreword to Dr. Smith's hook, says that in the next decade (1961-71), "The preservation [in deep freeze] of whole organs for trans- plantation may become possible . . ." (110)
Dr. Juan Negrin, Jr. (Lenox Hill Hospital, New York) is reported in 1961 as saying, "We are working now to develop a method for using full body freezing to suspend life. We have already succeeded in bringing about this state in various animals." (117) Some new successes will no doubt be on a cut-and-try, empirical basis. But in order to get a better idea of future prospects and present possibilities, let us briefly review current ideas about freezing damage.
The Mechanism of Freezing Damage
There are several suspected reasons for the frequent failure of animal cells and tissues to survive after being cooled to very low temperatures, stored, and thawed.
Before listing these possible causes of freezing damage, it should be pointed out that "failure to survive" is a very vague and possibly misleading expression. The usual criterion for survival is resumption of function, if an entire organ is involved, or growth in culture or successful transplant or autoreplant if a piece of tissue is in question. (Autoreplant refers to grafting the tissue back into the donor animal.) A tissue just below the borderline of resumed function is called "dead," and an experiment in which only a small percentage of the cells survive may be considered a failure. But in fact, near successes and partial successes afford substantial grounds for optimism, since they sug-
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gest that a comparatively small amount of damage has been done.
It is convenient to list several separate types of possible freezing injury, even though they are not all mutually exclusive, as follows:
1.There may be mechanical damage by ice crystals.
The most obvious opportunity for injury would be a stabbing, crushing, or bursting action against the cell membranes and cell bodies by the ice crystals formed as water freezes. Yet oddly enough, this kind of event seems rarely to have been observed, although it may sometimes occur. (In the case of plant tissues, with their more rigid membranes, this kind of damage occurs much more easily.)
In slow freezing-involving a typical cooling rate of, say, one Centigrade degree per minute-pure ice gradually separates out from the solution in the cell, the ice crystals forming beyond the membrane in the intercellular spaces. Slower freezing produces crystals that are larger in size, and of course fewer in number; faster freezing, the reverse. When the so-called eutectic temperature is reached, the remaining solution freezes out in a close mixture of crystals of ice and of the various salts or their hydrates.
There is ample evidence that ice crystal formation as such is not necessarily fatal, even though water expands when it freezes. Meryman says: "Experimental frostbite research produces evidence that a dog's leg can survive after the deep tissues have been at a temperature well below freezing for as much as fifteen to thirty minutes . . . There is no question but that ice crystals are formed, and yet the tissue survives . . . there appears to be little question but that in the soft tissues encountered in the animal kingdom it is possible for an ice crystal to intrude itself between the cells and to collapse the cells completely without impairing their capacity for survival." (70)
In fast freezing, the crystals formed are much smaller, and
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possibly for that reason less dangerous mechanically, even though the total volume of ice is the same. But fast freezing does not allow the water to leave the cell, and small intracellular or even intranuclear ice crystals may form, with poorly known but probably dangerous potentialities. For example, a membrane surrounding the cell nucleus may be violated.
2.There may be a dangerous concentration of electrolytes.
Since freezing involves a separation of ice from solution, it is a process of dehydration. The fluid left behind in the cell has an unnaturally high concentration of salts and similar substances, called "electrolytes," which have special electrical and chemical properties. This drastically changed internal environment may be fatal to the cell. (69)
Damage to the cell from this cause is thought to be dependent on the degree of electrolyte concentration, the time of exposure to it, and the temperature; a lower temperature means a slower reaction. The electrolyte concentration may be dangerously high, depending on the type of cell and other factors, roughly between 0C and -25C. Hence cooling in this range should be relatively rapid, if possible, in the absence of protective infusions.
Dr. J. E. Lovelock thinks the lipoproteins are especially sensitive to denaturation, or loss of chemical characteristics, from this cause. "A frequent if not invariable component of the many membranes of a complex living cell is the lipid-protein complex . . . held together not by the relatively strong covalent bonds which link the atoms of a simple protein, but by weak association forces similar to those supporting a soap bubble . . these complexes are inherently unstable and probably maintained in living cells by continuous synthesis . . . Freezing [can easily] denature the more sensitive lipid-protein complexes of the cell.
"The high sensitivity of lipid-protein complexes to the adverse effects of freezing suggests that not only the principal cell membrane, but also the lesser membranes of the cell . . . may suffer
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irreversible damage during freezing. The profound change in the environment of the cell which occurs during freezing is also capable of causing harm to the more stable molecular constituents of the cell." (62)
Not to put too fearful a face on it, we should note also that he goes on to say, ". . . many living cells and tissues have now been stored successfully in the frozen state ... In spite of these formidable hazards.
We should also remind ourselves once more that the phrase irreversible damage" is used much too cavalierly, and really means only "incapable of being reversed by methods so far employed."
3.There may be metabolic imbalance.
Dr. L. R. Rey, a prominent investigator of the Ecole Normale Superieure, Paris, believes the cells may be thrown out of kilter by the unequal effect of cold on delicately balanced life processes. Various enzymes are not inhibited in the same manner ... there may be an abnormal accumulation of intermediate metabolites which normally have a transitory existence and which may either prove to be toxic or to orient the metabolism in a different direction." (90)
This sounds rather hopeful, since it seems to leave open the possibility of redressing the balance, once we have both the understanding and the means.
A similar comment has been made by Dr. L. Kreyherg. "It is evident that in areas of organized tissue in situ [on site] the limits for survival of some of the cells after freezing . . . is not decided by the tolerance of the individual cells, but by the local reactions to the disorganization of the social life of the cells." (56) One suspects a like remark might apply to conditions within an individual cell and between its parts.
4.There may be thermal shock and osmotic shock.
Rapid freezing is fatal to many cells, for reasons not well un-
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derstood. One hypothesis about "thermal shock" is that various materials in the cells and their membranes shrink at different rates as the temperature is lowered, setting up destructive mechanical stresses. "Osmotic shock" refers to the unfavorable effects of sudden changes in solute concentrations in contact with certain membranes.
5.There may be damage during storage.
The cell encounters various vicissitudes as it is cooled, depending on many factors in each of several ranges of temperature; and when it finally arrives at storage temperature, its troubles may not be over. As already pointed out, there is evidence that at all but the very lowest temperatures, near absolute zero, eventually appreciable changes do take place, although they may be very slow.
Although Fernandez-Moran has pointed out that free radical activity can occur at - 196C, and suggested that perhaps long- term storage ought to be at liquid helium temperatures, nevertheless most writers seem to agree that storage at the temperature of boiling nitrogen is probably safe.
In any case, the word "decay" is probably ill chosen to describe the deterioration that may take place at low temperatures. It is probably not a case of general rot or putrefaction, or even normal metabolism, proceeding at a reduced rate, but rather a case of a few sensitive processes going essentially to completion, with ensuing stability for an indefinite period. If this is true, cooling with dry ice for long periods may be just about as safe as cooling with liquid helium, except for some initial minor damage. On this, however, I cannot quote authority, and many questions remain unanswered.
6.There may be thawing damage.
There is ample evidence that more damage may occur during thawing than during freezing, especially if thawing is slow and protective infusions lacking. The mechanisms of damage ap-
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pear to include migratory recrystallization of ice (small crystals may merge into larger crystals, causing mechanical disruption) and gas bubble formation, as well as others. These effects may occur at temperatures as low as -40C.
For a time, the difficulty of obtaining fast thawing was thought to be extremely serious for any but the smallest specimens, for which heat exchange is not a problem. It now appears, however, that microwave diathermy and induction methods will allow rapid thawing, at a more or less uniform rate throughout the body, even of large specimens. These methods involve the use of high frequency radio waves, alternating magnetic fields, or alternating electric fields; the former are analogous to an ordinary h eating lamp, the latter to so called electronic ovens. Apparatus has been described by Lovelock. (61) Using this, rabbits can be thawed in just a few seconds. (110)
7.There may be miscellaneous deleterious effects.
Various bits of evidence and speculation point to additional possibilities in the complex question of freezing injury. Drugs and antibiotics, as well as normal body solutes, may become concentrated to lethal levels. At dry ice temperature, if glycerol is used, there may be incomplete freezing, and a slight solubility of salts in glycerol may allow slow damage. At extremely low temperatures, complete removal of water as ice might include water molecules necessary for the structural integrity of proteins. And so it goes; much is known, but much more needs to be learned.
In summary, if we seek the main danger to humans frozen without perfusion by protective chemicals, expert consensus seems to point to denaturation of protein molecules, a consequence of overexposure to concentrated salt solutions, which in turn is a consequence of too-slow freezing. As to the possibility of avoiding this danger by using protective agents, or by increasing the speed of freezing, more will be said later.
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We are now in a position to answer the skeptics who say that, because a frostbitten toe may be incurable today, they doubt it will ever be possible to freeze and revive a complete man; and it may be worth while to make the answer explicit.
To begin with, frostbite often is cured, as shown by both clinical and laboratory experience. When we investigate which cases are cured and which are not, we find some neat tie-ins with the earlier discussion of the mechanism of freezing damage.
It has been shown, both in man and other animals, that freezing may actually occur, with formation of ice crystals in the tis- sues, without any irreversible harm. (110) The damage occurs if the temperature is too low, so that too much ice separates out, producing too high a concentration of solutes in the tissue fluids; or if freezing is too protracted, resulting in exposure of the cells to concentrated solutes for too long a time; or if thawing is too slow, resulting in dangerous high-temperature exposure to somewhat concentrated solutes; or if there has been bending or rubbing of the member while frozen, damaging nonresilient tissues; or if unfrozen but chilled and malfunctioning blood vessels fail to supply the thawed parts.
Medical texts recognize that thawing should be rapid, and rubbing (with snow or anything else) avoided. (12)
In a word, the presently incurable cases of frostbite are simply those cases in which the conditions were unfavorable. In other cases, frostbite can be cured. In fact, human skin has been rap idly frozen to dry ice temperature, and then used in grafts with some success. (110) Rabbit skin has been stored at dry ice temperature for four years without deterioration, after being pre- treated with glycerol. (110)
It is not obvious how a whole man could be rapidly frozen,
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or treated with glycerol, but these matters will be discussed later. The point here is simply that much is known about frostbite, it is preventable, and it is often curable. In addition, of course, some of the cases now thought incurable will be curable in the future.
The Action of Protective Agents
A brief review of the substances which have been found useful as protective infusions to prevent or reduce freezing damage, and of the theory of their action, shows that a good beginning has been made in the research, and that we are not without resources even now.
An ideal protective agent is one to which the cells are readily permeable, which prevents all kinds of freezing damage but is not itself toxic, and which can be easily removed after thawing. Nothing is known which completely fills this bill for all kinds of tissues. The substances which seem to be most nearly and most widely satisfactory are glycerol and dimethylsulfoxide.
Glycerol, in particular, has been extensively tested. Its use has been markedly successful, although not always completely successful, with a wide variety of organisms and tissues, including mammalian kidneys, bone, lungs, sperm, skin, hearts, ovarian and testicular tissue, and-most important-nervous tissue. (110)
In most cases, glycerol is thought to exert its beneficial action mainly by buffering the solution of electrolytes, that is by somehow preventing or reducing the chemical action of the dissolved substances. This action may be linked to the capacity of glycerol to bind water, and itself to dissolve some of the salts. Glycerol also suppresses the occurrence of a sharp eutectic point in physiological media; if there is no sudden crystallization, the cells may be saved from osmotic shock. (110) Other modes of protec-
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tion may also occur, and the relative importance of the various modes depends on the nature of the tissue.
Other substances, especially various sugars and alcohols, have been used with varying degrees of success.
Many fascinating experiments have been reported whereby tissues have been induced to tolerate glycerol as a result in adjustments of other components of the solution used for perfusion, such as calcium and potassium; and whereby ingenious methods of removing the glycerol have been devised. It is encouraging to note that in a great many cases where unsolved problems remain, it is the thawing phase and the removal of glycerol which seem to present the difficulty. This suggests that our bodies might be frozen and stored in reasonably good condition, so that future technicians would only have to perfect methods of thawing and removing the protective agents, and would not have to perform excessive wonders in reversing freezing damage.
The Persistence of Memory after Freezing
Some scientists not so long ago feared that even if we could freeze a body, store it at low temperatures and then restore it to active life, the brain would be wiped clean of memories, resulting in a kind of grown infant or idiot. It is obviously of the utmost importance to assure ourselves that this will not be the case.
Everything hinges on whether memory is dynamic or static. In computing machines, there are two general ways to store information: there are dynamic methods, involving oscillations which will die out if the power is turned off, and there are static methods, such as the use of magnetic tape, in which the information remains even though the machine is not turned on. These two possibilities exist for the brain as well.
As recently as 1960, Professor William Feindel of McGill
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University wrote: . . . nerve cells have some of their numerous branches turning hack to end. On the body of the parent cell, so that they actually receive samplings of their own outgoing messages . . . these self-re- exciting nerve loops may keep up a perpetual circular impulse which is the 'memory' of that particular cell .... (29) But he also pointed out that memories might be related to physical, chemical, or electrical changes at the hundreds of tiny button-like endings covering each nerve cell in the brain.
More recently, however, Professor IS. Roy John, director of the University of Rochester Center for Brain Research, has written: "Ample evidence exists for a two stage process of memory . . . (1) an early consolidation period approximately 0.5-1.0 hour long, in which reverberatory electrical activity probably maintains a representation of the experience, and (1) a long-lasting stable phase, in which experience is stored as a structural modification of some sort." (51)
In other words, very recent memories are dynamic, and this helps to explain the retrograde amnesias sometimes observed after certain kinds of shock or trauma. But most of the memories, the long-term memories, are static. In fact, they are believed to consist of changes in protein molecules in the brain cells. (46)
Many experimental tests have been made. For example, Dr. Smith reports, "We found, in collaboration with animal psychologists, that rats which had been trained to solve problems of finding food in mazes showed no appreciable loss of memory after cooling to a body temperature just above freezing. . . Activity of the cerebral cortex, as judged by electroencephalograms, ceases at about + 18C in the rat, so that cerebral activity most have been arrested for 1-2 hr. in all the animals tested. After reanimation they were, nevertheless, capable of acting on previous experience. This result was not consistent with the theory that memory depends upon a continuous pas-
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sage of nerve impulses through actively metabolizing neurons in the brain." (110)
There are two other points of great significance concerning memories: each one seems to be stored in many separate locations in the brain, and therefore may withstand widespread damage; and they consist of chemical coding similar to the traces which record genetic and immunological information, and possibly therefore they may be hardy and resistant to dam- age.
Professor Hans-Lukas Teuber of the M.I.T. writes, "Experiments employing massive cerebral ablations [removal of parts] or multiple transections [cross sections] of cortex . . . show remarkable resiliency of established 'engrams.' . . . The survival of old established traces following hibernation, general anesthesia, or convulsions suggests a mechanism protected against loss in a manner analogous to immune reactions, i.e., by virtue of multiplication of the trace, relatively small size, and considerable dispersal throughout the cerebrum. . . . [Certain experiments may reveal] that biological trace processes are of essentially the same type, whether we are dealing with genetic processes, embryonic induction, with learning, or immunological.(116)
We shall see the importance of this view when we ask how much freezing damage may be tolerable.
It must be emphasized that freezing damage, especially to the brain, may not be excessive, even though no mammal has yet made a complete recovery after full-body freezing by the rather crude methods so far employed.
There are several difficulties in freezing large animals. Perfusion with protective agents is not easy, and fast freezing of
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deep tissues has been regarded as hopeless. It follows that there may be denaturation of protein molecules in the brain by concentrated salts, and this thought has produced much gloom. In the next section it will be suggested that the major part of the freezing damage can, in fact, be avoided. In this section it will be argued that, even if the freezing injury is as severe as it usually seems to be, reasonable grounds for optimism remain.
First, while it may be hard to conceive of a generalized method for reversing protein denaturation, this is by no means the end of the story. For one thing, such a method may very well be devised, despite our inability to conceive it, by the ingenious men and redoubtable machines of the future. After all, in the last century engineers considered a heavier-than-air flying machine impossible; and before 1926, when Sumner isolated urease, it was not even known for sure that enzymes were proteins. (3) Further, as we shall see, the nature and extent of the denaturation is not uniform, and may in some cases be trivial; and the attack need not necessarily be "generalized."
It must be stressed that even crude freezing frequently fails to kill all cells, and that those "killed" suffer varying degrees of damage; this is true even if we fix our attention on a single type of tissue. Also, the most important parts of the cells may be the hardiest.
That some cells survive freezing even when most "die" we note, e.g., from the work of Rey, who rapidly cooled embryo chicken heart tissue: " . . . there is no growth in the cultures without glycerol except sometimes in two or three migrating cells . . . some peculiar cells do survive after the exposure to liquid nitrogen. . . . Why is the main part of the tissue killed by rapid cooling in liquid nitrogen? . . . we think [these alterations] occur during the thawing process." (90)
Even though chickens are not people and hearts are not brains, it is important that some cells survive; we can logically
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conclude that probably many others almost survived, and could have been rescued by future scientists either before or after thawing by improved methods.
By way of analogy, imagine viewing (from the air) a strafing attack on a column of troops. If none gets up afterwards, perhaps they are all dead. But if even one or two get up, it is highly probable that many others are merely wounded and not killed.
Again, Kreyberg says: "It is evident that through severe exposures to cold, many cells, sometimes most of the cells, succumb. Sometimes single cells, sometimes smaller groups of cells survive and are able to repopulate cultures and even form rather complex transplants, as demonstrated through the experiments with ovarian tissues." (56)
There is somewhat similar experience with mammalian nervous tissue, which is the most vital concern. Pascoe, working with rat ganglia, found that although one experiment was mainly negative, "One preparation [without glycerol] was stored overnight at - 150C and on warming the post-ganglionic nerve gave a small action potential when it was stimulated directly." (86)
Not only does experiment indicate that some cells survive unfavorable freezing methods, but theory also. The act of freezing will catch various cells in many different environmental situations and at different phases of the metabolic cycle. Some of these are almost sure to be lucky ones.
Further evidence that freezing damage to the brain may be only moderate, even in the absence of protective infusions, seems to be provided by the work of Dr. H. L. Rosomoff, of The Neurological Institute, New York. He produced lesions in dogs' brains by contact of the dura mater (brain integument) with a brass tube containing liquid nitrogen for eight minutes. If the dogs were kept at normal temperature afterwards, they invariably died, and microscopic examination showed "wide-
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spread destruction of cellular elements, especially the neurons, complete loss of cytoarchitectural markings, . . ." But of seven dogs kept at 25C or less (after the lesions were produced) for eighteen hours before rewarming, two survived, and the others lived five times longer than those not kept hypothermic; furthermore, examination of the lesions showed that: "The cortical architecture was better preserved, cellular elements showed less evidence of injury, albeit definite degenerative changes were found which may or may not have been reversible in nature." (93)
This experiment was not intended to study freezing damage as such, but was meant to investigate the benefit of hypothermia (reduced temperature) in the aftercare of any kind of brain lesion. Nevertheless, the damage to the cells in the lesion region presumably was produced by freezing. This seems to indicate rather clearly that the most serious damage following such freezing may be the result of anatomical and physiological events during and after thawing, and that while frozen the cells were in relatively good shape. As already pointed out, this is very important, since our task need only be to preserve the bodies with as little damage as may be; if necessary, we can leave to the future the problem of proper treatment during and after thawing.
Again, in the case of nervous tissue pre-treated with glycerol, there is evidence that the major difficulty may lie not in freezing and storage, but in the removal of glycerol. Dr. Smith, commenting on the work of Pascoe, who studied rat nervous tissue after perfusing whole rats with glycerol solution, says, . . damage to nervous tissue might not be a limiting factor in attempts to resuscitate a whole animal which had been perfused with glycerol and cooled to and thawed from a very low temperature. " (110)
Having gone to considerable pains to show that even crude
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freezing methods may not kill all cells, and that even many of the "nonsurvivors" may be only slightly damaged, we are now ready to make our conclusions more explicit.
It will be helpful if the reader will tentatively accept two propositions which will be given support in later chapters:
(1) Mastery is beginning to be obtained, and will eventually be thorough, over the growth, development, and differentiation or specialization of both genetic and somatic (body) cell& It will become possible to grow replacement parts, large or small, in culture, or alternatively to make the body repair itself by regenerating missing parts. (In the case of the brain, of course, there cannot be complete replacement or regeneration, since this would be equivalent to growing a new individual.)
(2) Wealth and resources will grow in the future at an ever increasing rate, qualitatively as well as quantitatively. In particular, there will be available fabulous machines, capable not only of action on a titanic scale but also of "thought" on extremely high levels and manipulation on microscopic levels. Now, we recall that memories are stored probably as changes in protein molecules in the brain cells, with multiple locations for each trace in many regions of the brain. (And since the memory recordings are thought to be chemically similar to the codings of genetic information, and since the latter is known to withstand liquid helium temperatures, it may be that memories are equally hardy, but we are not depending on this.) Other elements of personality may be represented in a similar way, or they may inhere in larger-scale circuitry, as in the fibre connections among the nerve cells.
There seems a good chance that the supra-molecular circuitry can be read well enough after freezing. Hence it may well be that only a small percentage of the brain cells need escape with little damage; this may be enough for reasonably faithful reconstruction of the brain with freshly generated tissue.
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The robot surgeons of the future will have powers now only faintly foreshadowed, but beginnings have already been made in cell surgery. Individual cells have been successfully operated upon, e.g., transplanting nuclei into enucleated amoebae, even cross-species! (27) Thus, if brute-force methods are necessary, it is not inconceivable that huge surgeon-machines, working twenty-four hours a day for decades or even centuries, will tenderly restore the frozen brains, cell by cell, or even molecule by molecule in critical areas.
We hasten to add that in all likelihood the methods used will be much more elegant and yet unforeseen. The great chemist, Linus Pauling, speaking in a general sense not so long ago, said, "The great discoveries of the future-those that will make the world different from the present world-are the discoveries that no one has yet thought about. . . . I know . . . that . . . discoveries will be made that I have not the imagination to describe-and I am awaiting them full of curiosity and enthusiasm." (88)
We must also bear prominently in mind that only those frozen in the very near future may be severely damaged; there will soon be accelerated research, and before many years non- damaging techniques should be available. Indeed, a man can probably be frozen right now with comparatively little injury, as we shall see in the next section.
Rapid Freezing and Perfusion Possibilities
Is a high freezing rate, with cooling of many degrees per minute, really out of the question for an animal as massive as man? And what are the chances of giving a complete large organism the protection of perfusion with a protective agent like glycerol?
It seems that in the absence of a protective agent, the brain (and body) should be frozen quickly. This will not prevent all
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damage, but might reduce the major risk of protein denaturation. How may quick freezing be accomplished?
Merely immersing the head or body, or even the naked brain in a cold bath such as liquid nitrogen will not do it, except for the outer layers. And while methods of heat transfer other than simple conduction do exist, they do not now seem applicable to cooling the body. The only means that seem presently feasible require more brain surface in contact with the refrigerant.
The most obvious method would be to circulate cold fluids through the brain's blood vessels. This in fact is done in open-heart surgery, but at temperatures above freezing. Whether anything could be done in the subzero range is, so far as I know, one of the open questions requiring investigation. It would certainly be difficult, with vessels tending to be brittle and clogged as well as constricted, but it is not obviously impossible.
Certain heroic measures also suggest themselves. For example, the brain might be "teased" apart into smaller segments which could be cooled more quickly. Or hollow needles carrying refrigerant might be inserted, as into a pincushion; care would be taken to penetrate different regions in the two hemispheres, to avoid destroying the homologous tissues on each side. Or the brain, after cooling, might even be sliced into sections for quick freezing, on the theory that this mechanical damage, although massive by present criteria, might yet be small compared with the damage done by slow freezing, and more easily repaired.
But the method of choice at the present time would seem to be moderately slow freezing after perfusion with glycerol solution.
Apparently there have been relatively few attempts at full- body perfusion. Dr. Smith says, "So far no technique has been evolved for perfusing individual organs or the whole mammal with glycerol and removing it without damage. If this could be done it might be possible to cool the intact mammal to and re-
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suscitate it from temperatures as low as -70C. Long-term storage of frozen mammals might then be considered. It must be emphasized that there is no prospect of accomplishing this in the near future." (110)
The great thing, however, is that we do not need the fullness of this accomplishment in the near future! Whole rats have been perfused, as previously noted, and probably men could be also. The problem of removing the glycerol without damage can be left to the more distant future, along with the problem of repair of those parts not reached or incompletely protected by the glycerol. The people who are dying right now cannot, and need not, wait for 100 per cent mastery of the problem.
The Limits of Delay in Treatment
If you have a dying relative, you can probably give him his best chance by obtaining skilled medical help, planned in advance, to prepare, perfuse, and freeze the body. If this kind of help is not available, and you nevertheless want to give him some chance, more desperate measures are required. In any case, it is important to know how soon after death treatment must be started, and this question will now be considered.
Many laymen, and even many physicians, have the impression that the body must be frozen within a few minutes after clinical death in order to have a chance of revival. This is an error.
It is quite true that if the oxygen supply is cut off, the brain ordinarily seems to suffer damage within three to eight minutes. But this seemingly simple statement is very deceptive: the words "ordinarily" and "damage" beth require clarification.
If death comes unexpectedly or without preparation, the brain may certainly suffer "irreversible" damage. When the blood circulation stops, there is no more delivery of oxygen and dextrose,
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and no more removal of waste products. The immediate causes of damage, according to Wolfe, include increase in inter- and/or intracellular fluid, loss of tone in the capillaries, increased permeability of tissues lining the blood vessels, disturbance of fluid balance, and concentration of lactic acid. (129)
How quickly the damage begins to occur is not entirely clear. Total circulatory interruption is considered dangerous after three minutes, and the most commonly mentioned limit of tolerance of the brain to lack of oxygen is perhaps five minutes. But Brockman and Jude have conducted experiments with dogs indicating that ten minutes of oxygen deprivation causes no harmful effects, although fourteen minutes at normal body temperature is fatal. They believe the shorter estimates result from use of methods which leave circulation depressed after the period of anoxia, producing added damage and causing the experiments to be misinterpreted. (10)
Of course much depends on temperature and individual variation, as well as other factors. In a later chapter we shall recount the story of a boy who made a nearly complete recovery after twenty-two minutes under water and 2, 1/2 hours of clinical death.
While the brain cells do indeed "die" more quickly than cells of other kinds, we must not therefore come to a hasty pessimistic conclusion. As already indicated, it may well be that the most important parts and functions of these cells are not so delicate as the cell as a whole.
By way of crude analogy-which of course must not be stretched too far-consider a bicycle and a huge snowball rolling down a slope. The bicycle is much more complex, and it can be stopped by merely thrusting a stick through the spokes, while much more effort is needed to stop the snowball. Just the same, a bicycle is on the whole much sturdier than a snowball, and when the stick is removed it will be ready to roll again.
It is possible, then, that hope should not be given up so long
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as any of the body cells show life. If the skin, for example, is still alive, then there is some chance that the brain cells are also alive, albeit damaged. Removal of excess lactic acid, adjustment of the fluid balance, and so on, by techniques at the disposal of future science, may find them good as new.
The period of grace before all of the body cells die is measured at least in hours, and perhaps in days. According to Lillehei et al., the stomach remains alive and healthy outside the body, even without cooling, for at least two hours. (59) Gresham, referring to an unpublished study by V. P. Perry, says, "Tissues removed from cadavers as late as 48 hr postmortem have, in most instances, shown cellular outgrowth in tissue culture. Although this does not eliminate the possibility of cellular alteration, it suggests that many tissues may remain functional for relatively long periods after death, and that postmortem tissues may be satisfactory for viable grafting." (36)
Boiling all this down to a rule of thumb, what perhaps emerges is that in a sell-reliance situation, if you want to give the deceased the benefit of even a relatively slim chance, a body should be frozen if it is found on the day of death. If the body has been exposed to cold weather, perhaps the chances are not too remote after two days. It seems entirely possible that the de- lay damage will still be no greater than the damage of the crude freezing method you may have to use.
In a hospital situation, with medical cooperation, the story is different and much more hopeful, and further remarks are called for.
The Limits of Delay in Cooling and Freezing
Three separate phases of postmortem care of the body may be distinguished: measures in advance of cooling, cooling down to the freezing range, and cooling down to storage temperature.
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For various reasons, death may come before cooling equipment is ready. Looking for means to prevent deterioration meanwhile, we find interesting possibilities. Some of them are relevant only if specialized equipment and personnel are at hand, while other measures can be employed by almost anyone.
Methods are already being applied to keep freshly dead bodies in good condition, for the purpose of maintaining organs in good health, when a transplant is contemplated but can- not be performed immediately. Heart-lung machines have been used to keep the body supplied with oxygenated blood for up to eighteen hours after death, and then livers taken from the bodies and used for grafts. (Detroit Free Press, October 31, 1963.)
An obvious resort in emergency is to use artificial respiration and external heart massage. (At the same time, the body could be cooled with ice packs, or by exposure to cold air.) Anyone can learn the techniques, and tubes are available so that mouth-to-mouth artificial respiration can be given without actual con- tact. Effectiveness would depend strongly on the cause of death and the condition of the body, but in some cases these simple measures might keep a body in reasonably good condition for hours. In other cases supporting measures might be needed, possibly including injection of anticoagulants.
In certain types of chest injury, possibly help might be obtained from a technique developed by Neely and coworkers. They perfused dogs with a buffered glucose solution instead of blood, and found that ". . . animals can survive 30 min of asanguineous perfusion with no oxygen, and that the survivors exhibited no gross brain damage." (80)
Another intriguing possibility, if the equipment were available, is suggested by the work of Dr. I. Boerema of the University of Amsterdam, The Netherlands. He has obtained some remarkable results treating patients inside a pressure caisson; the surgeons and attendants breathe air at three atmospheres pressure,
36
while the patient breathes pure oxygen at the same pressure. It has been found that the blood circulation can be arrested without harm for about twice as long as normal; at 14.5 deg C dogs can be kept a half hour or longer without extracorporeal circulation. Animals can actually live without blood; with pigs, the hemoglobin could be reduced virtually to zero for at least fifteen minutes, dissolved oxygen taking the place of oxygen carried by red corpuscles.
"... when an animal or patient breathes pure oxygen at 3 atmospheres (absolute) there is a greatly increased physical solution of oxygen in all tissues of the body, both fluid and semi- fluid. . . . [There is] extreme saturation of the whole body with physically dissolved oxygen, so that the cells have a much higher reserve of oxygen than they normally have. . . . We may assume, then, that the increased amount of oxygen in solution provides a true reserve for the tissues and, consequently, that the t issue cells can withstand a circulatory arrest of longer duration." (7)
If a terminal patient could be kept in such a chamber, there would be a wider margin of safety when he died. Or if a newly dead patient were put in such a chamber, artificial respiration and heart massage might work more effectively.
With fully adequate preparation, equipment, and personnel, the cooling phase seems to present little problem in most cases. Heartlung machines and heat exchangers are available at many hospitals. The cardiopulmonary bypass technique is commonly used for open-heart surgery, with cooling of the blood and body from the normal of about 38C down to 20C, and sometimes lower; this technique has been described, for example, by Sealy and coworkers. (104) Apparently it could also be used, depending on the cause of death and opportunity for preparation, to cool freshly dead bodies quickly and safely, with no damage to the brain.
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Finally, we ask how long it is safe to keep the body, after it has been cooled and before it has been frozen.
If a heartlung machine has been used, and continues to be used, this time may be more or less indefinite.
If the brain has reached the vicinity of 10C without damage, for example by use of a heartlung machine which then has to be disconnected, it can survive up to an hour without blood circulation, although there may be some relatively minor damage, if use is made of carotid arterial infusion of low molecular weight dextran; this is on the basis of experiments of Edmunds and co-workers with living dogs. (25)
Likewise the experience of Egerton and coworkers with patients undergoing hypothermic open-heart surgery showed that temperatures below 12C for more than forty-five minutes produced some brain damage, although most of them made complete recovery within four months. (26) Other work also shows that there is some brain damage, even if the blood still circulates, when the temperature reaches the vicinity of 0C, the freezing point of water.
Hence probably the body should not be cooled below about 10C before the freezing equipment is made ready, if this can be done within an hour or so later, as ought to be possible in a hospital.
Maximum and Optimum Storage Temperature
There seem to be four main possibilities for choice of storage temperature, and we must consider the theoretical and practical advantages and disadvantages of each. These are naturally occurring low temperatures in the arctic and antarctic, and the temperatures respectively of solid carbon dioxide, liquid nitro gen, and liquid helium.
By way of general introduction, Dr. Audrey U. Smith says:
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"The basic principle in storing living cells is to arrest the processes of aging and degeneration. When living cells are cooled there is a slowing down of the biochemical processes involved in respiration, metabolism and all the other interactions between the cytoplasm of the cells and their environment. If they are cooled to temperatures in the range below -79C in which carbon dioxide and other gases are solidified or liquefied, all chemical changes must either be slowed to a minute fraction of the normal rate or else completely halted. Aging should not occur and it should be possible to preserve them for infinitely long periods in this temperature range." (110)
Of course, "infinitely long" is a slight exaggeration, and in fact we know that some kinds of cells stored at dry ice temperature, -79C, do show slow changes, with the percentage of living (revivable) cells decreasing week by week or even day by day, even though other kinds of cells have shown no appreciable deterioration after several years. For example, Meryman says: "In the case of blood frozen without glycerol significant decay is measured in days of storage at -70C, weeks at -80C, months at -90 C, and years at -100C (70)
This does not necessarily mean that the relatively high temperatures are altogether hopeless. Some changes may take place, but little can yet be said about their extent and their reversibility. It may be that the changes, even though "fatal" by present tests, are minor, limited, and eventually reversible. It is not a case of general rot proceeding inexorably, although slowly; rather, it may be a case of some kinds of action not being completely inhibited, and stability may be reached after only some changes which, seen in perspective, are trivial.
Thus we cannot dismiss out of hand the suggestion sometimes heard that bodies be submitted to natural cold storage in arctic regions below the frost line. It has the obvious advantages of requiring no expensive investment and servicing, and of re-
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duced vulnerability in event of war. However, the coldest natural temperature is well above that of dry ice, and probably too high. The odds would seem heavily adverse.
For extremely long-term storage, there seems to be nearly- but not quite-universal agreement that liquid helium temperatures, in the neighbourhood of -270C, are safest . One of the dissenters is Dr. R. B. Gresham, who says: "It has been shown that after materials are frozen, there occurs a continuous thermodynamic activity down to -196C (-321F) or liquid nitrogen temperature, where movement ceases only to be noted again at -269C (-449F) or liquid helium temperatures. . . . Although the effects of this thermodynamic activity on long-term storage of living cells is not known, when storage time is to be measured in years, it is theoretically desirable to maintain a temperature of - 196C." (36)
This argument does not really seem very impressive. The "thermodynamic activity" and "movement refer merely to certain irregularities in the rate of heat loss as the temperature is lowered, and accompanying shifts in the molecular structure or physical state of materials, mainly water. As far as I can see, there is no particular reason to think this implies any instability at a fixed temperature, in general. Most writers do not seem to be worried by this question.
A more serious objection to use of the lowest temperatures is that while nothing will happen after storage temperature is reached, changes may take place en route. In other words, we should not use a temperature any lower than necessary, because we may be letting ourselves in for gratuitous trouble. In every range, more cooling means more change, and unnecessary changes are to be avoided.
On a practical level, liquid helium is relatively expensive, and tricky to handle.
What would seem to emerge, then, is the following. At the
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present time, the temperature of choice is that of liquid nitrogen. When permanent installations are built, probably liquid helium will be used. As an emergency or austerity measure, one might use dry ice, which is cheap and easy to handle.
Would a body in cold storage, although preserved against decay, be gradually "cooked" by the slow but inexorable attack of natural radiations?
We know these are all around us: cosmic rays bombard us from the skies; uranium, thorium, and radium in rocks and soil, in concrete and brick, spray penetrating emanations similar to X rays; and certain radioactive atoms (radioisotopes) in our own bodies dribble slow poison. (In addition to this natural "background" radiation, there is fallout radiation from testing of nuclear weapons, but this so far is more or less negligible.)
Since these radiations are of low intensity, they produce only a "chronic dose" which is scarcely noticed, since a functioning body can repair most of the damage as fast as it occurs. But all doses absorbed by a body in cold storage must be regarded as acute; we must consider the possibility that the cumulative dam- age to a frozen body might become serious as the centuries passed.
Examining the data, we find there may indeed be a problem, but not one too formidable. (The pertinent information can be found, for example, in The Effects of Nuclear Weapons, U. S. Atomic Energy Commission, 1962.)
The unit usually used to measure dosage of radiation is the "rem" (roentgen equivalent, mammal, or man); we do not need its technical definition, but may note that an acute dose of 100 rems or less is unlikely to produce noticeable illness, a dose of 600 rems results in severe radiation sickness requiring hospital-
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ization and the most competent care, and a dose of 1000 rems or more is almost certainly fatal with the present resources of medicine.
Background radiation varies considerably with locale, but as a rough average we might expect everyone to receive a dose of about 10 rems in 50 years. A stored body, then, might take 500 years to accumulate a "clinical" or symptom-producing dose of 100 rems, and 3,000 years to soak up a currently dangerous dose of 600 rems. These times, to be sure, might be reduced if nuclear war or excessive weapons testing produced heavy fallout; but they could also be much lengthened by precautions available at moderate expense.
If the bodies were stored underground, in vaults made of low-radioactivity materials, then they would be shielded from most of the external background radiation, leaving only the internal radiators to worry about. These consist mainly of one of the forms of the element potassium (the radioisotope potassium-40) found especially in the soft tissues of the body.
The dose rate due to potassium-40 is about 20 millirems (0.020 rem) yearly. This will continue essentially indefinitely, since the "half life," or time required for the dose rate to halve as the decaying potassium-40 is used up, is over a billion years. But to accumulate a dose of 100 rems would take 5,000 years, and for 600 rems the wait would be 30,000 years.
Even then, the radiation damage would no doubt be substantially less than the injury done (to the earliest frozen bodies) by crude freezing methods, so one might guess that at least 100,000 years must elapse before radiation damage becomes critical. I can think of certain heroic measures to extend this time to a million years or more, but it is hardly worth the trouble.
Most of us will be frozen by advanced methods developed in the next decade or two, and will be waiting in cold storage
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mainly for the solution of the aging problem. In light of the explosive acceleration of scientific progress, it would be astonishing if this were to take as long as 5,000 years. In this view, we can ignore the effects of bodily radiation damage.
However, a postscript may be worth while to reassure those worried about the genetic effects of radiation. It is true that a dose of 100-300 rems inflicted on everyone in every generation might eventually produce so many mutations or freaks of inheritance as to threaten the race, if nothing were done about it. But we expect eventually to control and tailor our genes, the physical blueprints of inheritance carried by our cells, and in any event the resurrected frozen will not constitute the entire populace. Individually, there is no cause for concern: a man exposed to 500 rems has only a negligible chance of observing deformities in his children or grandchildren. (See, for example, the article by Professor Muller in Radiation Biology, ed. Alexander Hollaender, McGraw-Hill, 1954.)
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