From: merkle@parc.xerox.com (Ralph Merkle)
Subject: The Technical Feasibility of Cryonics; Part #2
Organization: Xerox PARC
Date: 22 Nov 92 21:14:47 GMT

The Technical Feasibility of Cryonics

PART 2 of 5.

by

Ralph C. Merkle
Xerox PARC
3333 Coyote Hill Road
Palo Alto, CA 94304
merkle@xerox.com

A shorter version of this article appeared in:
Medical Hypotheses (1992)  39, pages 6-16.


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CRITERIA OF DEATH

"death \'deth\ n [ME deeth, fr. OE death; akin to ON dauthi death, 
deyja to die - more at DIE]  1: a permanent cessation of all vital 
functions : the end of life"
          Webster's New Collegiate Dictionary

Determining when "permanent cessation of all vital functions" has 
occurred is not easy.  Historically, premature declarations of death and 
subsequent burial alive have been a major problem.   In the seventh 
century, Celsus wrote "... Democritus, a man of well merited celebrity, 
has asserted that there are in reality, no characteristics of death 
sufficently certain for physicians to rely upon."[87, page 166].

Montgomery, reporting on the evacuation of the Fort Randall Cemetery, 
states that nearly two percent of those exhumed were buried alive[87]. 

"Many people in the nineteenth century, alarmed by the prevalence of 
premature burial, requested, as part of the last offices, that wounds or 
mutilations be made to assure that they would not awaken ...  embalming 
received a considerable impetus from the fear of premature burial."[87].

     New Criteria

Current criteria of "death" are sufficient to insure that spontaneous 
recovery in the mortuary or later is a rare occurence.  When examined 
closely, however,  such criteria are simply a codified summary of 
symptoms that have proven resistant to treatment by available 
techniques.  Historically, they derive from the fear that the patient 
will spontaneously recover in the morgue or crypt.  There is no 
underlying theoretical structure to support them, only a continued 
accumulation of ad hoc procedures supported by empirical evidence.  Each 
new medical advance forces a reexamination and possible change of the 
existing ad hoc criteria.  The criteria used by the clinician today to 
determine "death" are dramatically different from the criteria used 100 
years ago, and have changed more subtly but no less surely in the last 
decade[ft. 7].  It seems almost inevitable that the criteria used 200 
years from now will differ dramatically from the criteria commonly 
employed today.

These ever shifting criteria for "death" raise an obvious question:  is 
there a definition which will not change with advances in technology?  A 
definition which does have a theoretical underpinning and is not 
dependent on the technology of the day?

The answer arises from the confluence and synthesis of many lines of 
work, ranging from information theory, neuroscience, physics, 
biochemistry and computer science to the philosophy of the mind and the 
evolving criteria historically used to define death.

When someone has suffered a loss of memory or mental function, we often 
say they "aren't themselves."  As the loss becomes more serious and all 
higher mental functions are lost, we begin to use terms like "persistent 
vegetative state."  While we will often refrain from declaring such an 
individual "dead," this hesitation does not usually arise because we 
view their present state as "alive" but because there is still hope of 
recovery to a healthy state with memory and personality intact.  From a 
physical point of view we believe there is a chance that their memories 
and personalities are still present within the physical structure of the 
brain, even though their behavior does not provide direct evidence for 
this.  If we could reliably determine that the physical structures 
encoding memory and personality had in fact been destroyed, then we we 
would abandon hope and declare the person dead.

     The Information Theoretic Criterion of Death

Clearly, if we knew the coordinates of each and every atom in a person's 
brain then we would (at least in principle) be in a position to 
determine with absolute finality whether their memories and personality 
had been destroyed in the information theoretic sense, or whether their 
memories and personality were preserved but could not, for some reason, 
be expressed.  If such final destruction had taken place, then there 
would be little reason for hope.  If such destruction had not taken 
place, then it would in principle be possible for a sufficiently 
advanced technology to restore the person to a fully functional and 
healthy state with their memories and personality intact.

Considerations like this lead to the information theoretic criterion of 
death[ft. 8].  A person is dead according to the information theoretic 
criterion if their memories, personality, hopes, dreams, etc. have been 
destroyed in the information theoretic sense.  That is, if the 
structures in the brain that encode memory and personality have been so 
disrupted that it is no longer possible in principle to restore them to 
an appropriate functional state then the person is dead.  If the 
structures that encode memory and personality are sufficiently intact 
that inference of the memory and personality are feasible in principle, 
and therefore restoration to an appropriate functional state is likewise 
feasible in principle, then the person is not dead.

A simple example from computer science is in order.  If a computer is 
fully functional, then its memory and "personality" are completely 
intact.  If we took an axe to the CPU, then the computer would no longer 
be functional.  However, its memory and "personality" would still be 
present on disk, and once we repaired the CPU we could fully restore the 
computer[ft. 9].

In a similar fashion, as long as the structures that encode the memory 
and personality of a human being have not been irretrievably "erased" 
(to use computer jargon) then restoration to a fully functional state 
with memory and personality intact is in principle feasible.  Any 
technology independent definition of "death" should conclude that such a 
person is not dead, for a sufficiently advanced technology could restore 
the person to a healthy state.

On the flip side of the coin, if the structures encoding memory and 
personality have suffered sufficient damage to obliterate them beyond 
recognition, then death by the information theoretic criterion has 
occurred.  An effective method of insuring such destruction is to burn 
the structure and stir the ashes.  This is commonly employed to insure 
the destruction of classified documents.  Under the name of "cremation" 
it is also employed on human beings and is sufficient to insure that 
death by the information theoretic criterion takes place.

     More Exotic Approaches

It is not obvious that the preservation of life requires the physical 
repair or even the preservation of the brain[11,12].  Although the brain 
is made of neurons, synapses, protoplasm, DNA and the like; most modern 
philosophers of consciousness view these details as no more significant 
than hair color or clothing style.   Three samples follow.

The ethicist and prolific author Robert Veatch said, in "Death, Dying, 
and the Biological Revolution", "An 'artificial brain' is not possible 
at present, but a walking, talking, thinking individual who had one 
would certainly be considered living."[15, page 23].

The noted philosopher of consciousness Paul Churchland said, in "Matter 
and Consciousness," "If machines do come to simulate all of our internal 
cognitive activities, to the last computational detail, to deny them the 
status of genuine persons would be nothing but a new form of 
racism."[12, page 120].

Hans Moravec, renowned roboticist and Director of the Mobile Robot Lab 
at Carnegie Mellon said, "Body-identity assumes that a person is defined 
by the stuff of which a human body is made.  Only by maintaining 
continuity of body stuff can we  preserve an individual person.  
Pattern-identity, conversely, defines the essence of a person, say 
myself, as the pattern and the process going on in my head and body, not 
the machinery supporting that process.  If the process is preserved, I 
am preserved.  The rest is mere jelly."[50, page 117].

     We'll Use the Conservative Approach

Restoration of the existing structure will be more difficult than 
building an artifical brain (particularly if the restoration is down to 
the molecular level). Despite this, we will examine the technically more 
exacting problem of restoration because it is more generally acceptable.  
Most people accept the idea that restoring the brain to a healthy state 
in a healthy body is a desirable objective.  A range of increasingly 
less restrictive objectives (as described) are possible.  To the extent 
that more relaxed criteria are acceptable, the technical problems are 
much less demanding.  By deliberately adopting such a conservative 
position, we lay ourselves open to the valid criticism that the methods 
described here will not prove necessary.  Simpler techniques that relax 
to some degree the philosophical constraints we have imposed might well 
be adopted in practice.  In this paper we will eschew the more exotic 
possibilities (without, however, adopting any position on their 
desirability).

Another issue is not so much philosophical as emotional.  Major surgery 
is not a pretty sight.  There are few people who can watch a surgeon cut 
through living tissue with equanimity.  In a heart transplant, for 
example, surgeons cut open the chest of a dying patient to rip out their 
dying heart, cut open a fresh cadaver to seize its still-beating heart, 
and then stitch the cadaver's heart into the dying patients chest.  
Despite this (which would have been condemned in the middle ages as the 
blackest of black magic), we cheer the patient's return to health and 
are thankful that we live in an era when medicine can save lives that 
were formerly lost.

The mechanics of examining and repairing the human brain, possibly down 
to the level of individual molecules, might not be the best topic for 
after dinner conversation.  While the details will vary depending on the 
specific method used, this could also be described by lurid language 
that failed to capture the central issue:  the restoration to full 
health of a human being.

A final issue that should be addressed is that of changes introduced by 
the process of restoration itself.  The exact nature and extent of these 
changes will vary with the specific method.  Current surgical 
techniques, for example, result in substantial tissue changes.  
Scarring, permanent implants, prosthetics, etc. are among the more 
benign outcomes.  In general, methods based on a sophisticated ability 
to rearrange atomic structure should result in minimal undesired 
alterations to the tissue.

"Minimal changes" does not mean "no changes."  A modest amount of change 
in molecular structure, whatever technique is used, is both unavoidable 
and insignificant.  The molecular structure of the human brain is in a 
constant state of change during life - molecules are synthesized, 
utilized, and catabolized in a continuous cycle.  Cells continuously 
undergo slight changes in morphology.  Cells also make small errors in 
building their own parts.  For example, ribosomes make errors when they 
build proteins.  About one amino acid in every 10,000 added to a growing 
polypeptide chain by a ribosome is incorrect[14, page 383].  Changes and 
errors of a similar magnitude introduced by the process of restoration 
can reasonably be neglected.


     Does the Information Theoretic Criterion Matter?

It is normally a matter of small concern whether a physician of 2190 
would or would not concur with the diagnosis of "death" by a 
contemporary physician applied to a specific patient in 1990.  A 
physician of today who found himself in 1790 would be able to do little 
for a patient whose heart had stopped, even though he knew 
intellectually that an intensive care unit would likely be able to save 
the patients life.  Intensive care units were simply not available in 
1790, no matter what the physician knew was possible.  So, too, with the 
physician of today when informed that a physician 200 years hence could 
save the life of the patient that he has just pronounced "dead."   There 
is nothing he can do, for he can only apply the technologies of today - 
except in the case of cryonic suspension.

In this one instance, we must ask not whether the person is dead by 
today's (clearly technology dependent) criteria, but whether the person 
is dead by all future criteria.  In short, we must ask whether death by 
the information theoretic criterion has taken place.  If it has not, 
then cryonic suspension is a reasonable (and indeed life saving) course 
of action.


Experimental Proof or Disproof of Cryonics

It is often said that "cryonics is freezing the dead."  It is more 
accurate to say that "cryonics is freezing the terminally ill.  Whether 
or not they are dead remains to be seen."

The scientifically correct experiment to verify that cryonics works (or 
demonstrate that it does not work) is quite easy to describe:

     1.)     Select N experimental subjects.
     2.)     Freeze them.
     3.)     Wait 200 years.
     4.)     See if the technology available 200 years from now can
             (or cannot) cure them.

The drawback of this experimental protocol is obvious:  we can't get the 
results for 200 years.  This problem is fundamental.  The use of future 
technology is an inherent part of cryonics.

This kind of problem is not unique to cryonics.  A new AIDS treatment 
might undergo clinical trials lasting a few years.  The ethical dilemma 
posed by the terminally ill AIDS patient who might be assisted by the 
experimental treatment is well known.  If the AIDS patient is given the 
treatement prior to completion of the clinical trials, it is possible 
that his situation could be made signficantly worse.  On the other hand, 
to deny a potentially life saving treatment to someone who will soon die 
anyway is ethically untenable.

In the case of cryonics this is not an interrim dilemma pending the 
(near term) outcome of clinical trials.  It is a dilemma inherent in the 
nature of the proposal.  Clinical trials, the bulwark of modern medical 
practice, are useless in resolving the effectiveness of cryonics in a 
timely fashion.

Further, cryonics (virtually by definition) is a procedure used only 
when the patient has exhausted all other available options.  In current 
practice the patient is suspended after legal death: the fear that the 
treatment might prove worse than the disease is absent.  Of course, 
suspension of the terminally ill patient somewhat before legal death has 
significant advantages.  For example, a patient suffering from a brain 
tumour might view suspension following the obliteration of his brain as 
significantly less desirable than suspension prior to such obliteration, 
even if the suspension occurred at a point in time when the patient was 
legally "alive."

In such a case, it is inappropriate to disregard or override the 
patient's own wishes.  To quote the American College of Physicians 
Ethics Manual, "Each patient is a free agent entitled to full 
explanation and full decision-making authority with regard to his 
medical care.  John Stuart Mill expressed it as:  'Over himself, his own 
body and mind, the individual is sovereign.'  The legal counterpart of 
patient autonomy is self-determination.  Both principles deny legitimacy 
to paternalism by stating unequivocally that, in the last analysis, the 
patient determines what is right for him."  "If the [terminally ill] 
patient is a mentally competent adult, he has the legal right to accept 
or refuse any form of treatment, and his wishes must be recognized and 
honored by his physician."[92]

If clinical trials cannot provide us with an answer, are there any other 
methods of evaluating the proposal?  Can we do more than say that (a) 
cryonic suspension can do no harm (in keeping with the Hippocratic 
oath), and (b) it has some difficult-to-define chance of doing good?

Failure Criteria

Trying to prove something false is often the simplest method of 
clarifying exactly what is required to make it true.  A consideration of 
the information theoretic criterion of death makes it clear that, from a 
technical point of view (ignoring various non-technical issues) there 
are two and only two ways in which cryonics can fail[ft. 10].

Cryonics will fail if:

(1)     Information theoretic death occurs prior to reaching liquid 
        nitrogen temperature[ft. 11].

(2)     Repair technology that is feasible in principle is never developed 
        and applied in practice, even after the passage of centuries.

The first failure criterion can only be considered against the 
background of current understanding of freezing damage, ischemic injury 
and mechanisms of memory and synaptic plasticity.  Whether or not memory 
and personality are destroyed in the information theoretic sense by 
freezing and the ischemic injury that might precede it can only be 
answered by considering both the physical nature of memory and the 
nature of the damage to which the brain is subjected before reaching the 
stability provided by storage in liquid nitrogen.  The following 
sections will therefore provide brief reviews of these subjects.

The second failure criterion is considered in the later sections on 
technical issues, which discuss in more detail how future technologies 
might be applied to the repair of frozen tissue.

As the reader will readily appreciate, the following reviews will 
consider only the most salient points that are of the greatest 
importance in determining overall feasibility.  They are necessarily too 
short to consider the topics in anything like full detail, but should 
provide sufficient information to give the reader an overview of the 
relevant issues.  References to further reading are provided 
throughout[ft. 12].


FREEZING DAMAGE

There is an extensive literature on the damage caused by both cooling 
and freezing to liquid nitrogen temperatures.  Some reviews are[5, 6, 
68, 70].  Scientific American had a recent and quite accessible 
article[57].  In this section, we briefly review the nature of such 
damage and consider whether it is likely to cause information theoretic 
death. Damage, per se, is not meaningful except to the extent that it 
obscures or obliterates the nature of the original structure.

While cooling tissue to around 0 degrees C creates a number of problems, the 
ability to cool mammals to this temperature or even slightly below (with 
no ice formation) using current methods followed by subsequent complete 
recovery[61, 62] shows that this problem can be controlled and is 
unlikely to cause information theoretic death.  We will, therefore, 
ignore the problems caused by such cooling.  This problem is discussed 
in [5] and elsewhere.

Further, some "freezing" damage in fact occurs upon re-warming.  Current 
work supports this idea because the precise method used to re-warm 
tissue can strongly affect the success or failure of present experiments 
even when freezing conditions are identical[5, 6].  If we presume that 
future repair methods avoid the step of re-warming the tissue prior to 
analysis and instead analyze the tissue directly in the frozen state 
then this source of damage will be eliminated.  Several current methods 
can be used to distinguish between damage that occurs during freezing 
and damage that occurs while thawing.  At present, it seems likely that 
some damage occurs during each process.  While significant damage does 
occur during slow freezing, it does not induce structural changes which 
obliterate the cell.

     Present Day Successes

Many types of tissue including human embryos, sperm, skin, bone, red and 
white blood cells, bone marrow, and others [5, 6, 59] have been frozen 
in liquid nitrogen, thawed, and have recovered.  This is not true of 
whole mammals[ft. 13].  The brain seems more resistant than most organs 
to freezing damage[58, 79].  Recovery of overall brain function 
following freezing to liquid nitrogen temperature has not been 
demonstrated, although recovery of unit level electrical activity 
following freezing to -60 degrees C has been demonstrated[79].

     Fractures

Perhaps the most dramatic injury caused by freezing is macroscopic 
fractures[56].  Tissue becomes extremely brittle at or below the "glass 
transition temperature" at about 140K.  Continued cooling to 77K (the 
temperature of liquid nitrogen) creates tensile stress in the glassy 
material.  This is exacerbated by the skull, which inhibits shrinkage of 
the cranial contents.  This stress causes readily evident macroscopic 
fractures in the tissue.

Fractures that occur below the glass transition temperature result in 
very little information loss.  While dramatic, this damage is unlikely 
to cause or contribute to information theoretic death.

     Ice

The damage most commonly associated with freezing is that caused by ice.  
Contrary to common belief, freezing does not cause cells to burst open 
like water pipes on a cold winter's day.  Quite the contrary, ice 
formation takes place outside the cells in the extracellular region.  
This is largely due to the presence of extracellular nucleating agents 
on which ice can form, and the comparative absence of intracellular 
nucleating agents.  Consequently the intracellular liquid supercools.

Extracellular ice formation causes an increase in the concentration of 
the extra-cellular solute, e.g., the chemicals in the extracellular 
liquid are increased in concentration by the decrease in available 
water.  The immediate effect of this increased extracellular 
concentration is to draw water out of the cells by osmosis.  Thus, 
freezing dehydrates cells.

Damage can be caused by the extracellular ice, by the increased 
concentration of solute, or by the reduced temperature itself.  All 
three mechanisms can play a role under appropriate conditions.

The damage caused by extracellular ice formation depends largely on the 
fraction of the initial liquid volume that is converted to ice[6, 57].  
(The initial liquid volume might include a significant amount of 
cryoprotectant as well as water).  When the fraction of the liquid 
volume converted to ice is small, damage is often reversible even by 
current techniques.  In many cases, conversion of significantly more 
than 40% of the liquid volume to ice is damaging[70, page 134; 71].  The 
brain is more resistant to such injury: conversion of up to 60% of the 
liquid volume in the brain to ice is associated with recovery of 
neuronal function[58, 62, 66, 82].  Storey and Storey said "If the cell 
volume falls below a critical minimum, then the bilayer of phospholipids 
in the membrane becomes so greatly compressed that its structure breaks 
down.  Membrane transport functions cannot be maintained, and breaks in 
the membrane spill cell contents and provide a gate for ice to propagate 
into the cell.  Most freeze-tolerant animals reach the critical minimum 
cell volume when about 65 percent of total body water is sequestered as 
ice."[57].

Appropriate treatment with cryoprotectants (in particular glycerol) 
prior to freezing will keep more than 40% of the liquid volume from 
being converted to ice even at liquid nitrogen temperatures.

Fahy has said "All of the postulated problems in cryobiology - cell 
packing [omitted reference], channel size constraints [omitted 
reference], optimal cooling rate differences for mixed cell populations 
[omitted reference], osmotically mediated injury[omitted references], 
and the rest - can be solved in principle by the selection of a 
sufficiently high concentration of cryoprotectant prior to freezing.  In 
the extreme case, all ice formation could be suppressed completely by 
using a concentration of agent sufficient to ensure vitrification of the 
biological system in question [omitted reference]"[73].  Unfortunately, 
a concentration of cryoprotectant sufficiently high to protect the 
system from all freezing injury would itself be injurious[73].  It 
should be possible to trade the mechanical injury caused by ice 
formation for the biochemical injury caused by the cryoprotectant, which 
is probably advantageous.  In some suspensions done by Alcor both venous 
and arterial glycerol concentrations have exceeded 6 molar.  If this 
concentration of cryoprotectant is also reaching the tissues, it should 
keep over 60% of the initial liquid volume from being converted to ice 
at liquid nitrogen temperatures.

     Concentration Effects

"Dehydration and concentration of solutes past some critical level may 
disrupt metabolism and denature cell proteins and macromolecular 
complexes"[70, page 125].  The functional losses caused by this 
mechanism seem unlikley to result in significant information loss.  One 
qualification to this conclusion is that cell membranes appear to be 
weakened by increased solute concentration[5, page 92].  To the extent 
that structural elements are weakened by increased solute concentrations 
the vulnerability of the cell to structural damage is increased.

     Denaturing

Finally, denaturing of proteins might occur at low temperature.  In this 
process the tertiary and perhaps even secondary structure of the protein 
might be disrupted leading to significant loss of protein function.  
However, the primary structure of the protein (the linear sequence of 
amino acids) is still intact and so inference of the correct functional 
state of the protein is in principle trivial.  Further, the extent of 
protein denaturation caused by freezing must necessarily be limited 
given the relatively wide range of tissues that have been successfully 
frozen and thawed.

     Intracellular Freezing

Intracellular freezing is another damaging event which might occur[6].  
If cooling is slow enough to allow the removal of most of the water from 
the cell's interior by osmosis, then the high concentration of solute 
will prevent the small amount of remaining water from freezing.  If 
cooling is too rapid, there will be insufficient time for the water 
within the cell to escape before it freezes.  In the latter case, the 
intracellular contents are supercooled and freezing is abrupt (the cell 
"flashes").  While this correlates with a failure to recover function[5, 
6, 68, 70] it is difficult to believe that flash freezing results in 
significant loss of information.

Intracellular freezing is largely irrelevant to cryonic suspensions 
because of the slow freezing rates dictated by the large mass of tissue 
being frozen.  Such freezing rates are too slow for intracellular 
freezing to occur except when membrane rupture allows extracellular ice 
to penetrate the intracellular region.  If the membrane does fail, one 
would expect the interior of the cell to "flash."

     Loss of Information versus Loss of Function

Spontaneous recovery of function following freezing to liquid nitrogen 
temperatures using the best currently available techniques appears 
unlikely for mammalian organs, including the brain.  Despite this, the 
level of structural preservation can be excellent.   The complexity of 
the systems that have been successfully frozen and rewarmed is 
remarkable, and supports the claim that good structural preservation is 
often achieved.  While spontaneous recovery of function by the human 
brain cannot be viewed as likely, the mechanisms of damage that have 
been postulated in the literature are sufficiently subtle that 
information loss is likely to be small;  that is, death by the 
information theoretic criterion is unlikely to have occurred.


ISCHEMIC INJURY AND PRE-SUSPENSION INJURY

Today, cryonic suspensions cannot be initiated until after legal death.  
Even operating under this constraint, it is often possible to initiate 
suspensions within two or three minutes following cessation of 
heartbeat. Future suspensions might eliminate delay entirely[ft. 14].  
However, delay is sometimes unavoidable[ft. 15].  The most significant 
type of damage that such delay causes is ischemic injury.

It should be emphasized that delay in initiating cryonic suspension is 
caused by the current social and legal context.  From a technical point 
of view it is usually relatively easy to initiate suspension without 
delay and without ischemia.  It is therefore incorrect for two reasons 
to argue that cryonic suspension must fail because it is initiated 
following legal death.  First, legal death and information theoretic 
death are logically distinct: information theoretic death will often 
occur well after legal death.  Second, a change in legal climate would 
permit suspensions to begin prior to legal death.  This would completely 
eliminate ischemia as a cause for concern in the feasibility of 
cryonics.

Broadly speaking, the structure of the human brain remains intact for 
several hours or more following the cessation of blood flow, or 
ischemia.  The tissue changes that occur subsequent to ischemia have 
been well studied.  There have also been studies of the "postmortem" 
changes that occur in tissue.  Perhaps the most interesting of these 
studies was conducted by Kalimo et. al.[65].

     "Postmortem" Changes in the Human Brain

In order to study immediate "postmortem" changes, Kalimo et. al. 
perfused the brains of 5 patients with aldeyhydes within half an hour of 
"clinical death".  Subsequent examination of the preserved brain tissue 
with both light and electron microscopy showed the level of structural 
preservation.  In two cases, the changes described were consistent with 
approximately one to two hours of ischemic injury.  (Ischemic injury 
often begins prior to declaration of "clinical death", hence the 
apparently longer ischemic period compared with the interval following 
declaration of death and prior to perfusion of fixative).  Physical 
preservation of cellular structure and ultrastructure was excellent.  It 
is difficult to avoid the conclusion that information loss was 
negligible in these cases.  In two other cases, elevated 
intraparenchymal pressure prevented perfusion with the preservative, 
thus preventing examination of the tissue.  Without such an examination, 
it is difficult to draw conclusions about the extent of information 
loss.  In the final case, "...the most obvious abnormality was the 
replacement of approximately four-fifths of the parenchyma of the brain 
by a fluid-containing cavity that was lined by what seemed to be very 
thin remnants of the cerebral cortex."  Cryonic suspension in this last 
case would not be productive.

As an aside, the vascular perfusion of chemical fixatives to improve 
stability of tissue structures prior to perfusion with cryoprotectants 
and subsequent storage in liquid nitrogen would seem to offer 
significant advantages.  The main issue that would require resolution 
prior to such use is the risk that fixation might obstruct circulation, 
thus impeding subsequent perfusion with cryoprotectants.  Other than 
this risk, the use of chemical fixatives (such as aldehydes and in 
particular glutaraldehyde) would reliably improve structural 
preservation and would be effective at halting almost all deterioration 
within minutes of perfusion[67].  The utility of chemical preservation 
has been discussed by Drexler[1] and by Olson[90], among others.

     Ischemia

The events following ischemia have been reasonably well characterized.  
Following experimental induction of ischemia in cats, Kalimo et. al.[74] 
found "The resulting cellular alterations were homogeneous and uniform 
throughout the entire brain:  they included early chromatin clumping, 
gradally increasing electron lucency of the cell sap, distention of 
endoplasmic reticulum and Golgi cisternae, transient mitochondrial 
condensation followed by swelling and appearance of flocculent 
densities, and dispersion of ribosomal rosettes."  Energy levels within 
the cell drop sharply within a few minutes of cessation of blood flow.  
The chromatin clumping is a reversible early change.  The loss of energy 
results fairly quickly in failure to maintain trans-membrane 
concentration gradients (for example the Na+K+ pump stops working, 
resulting in increased intracellular Na+ and increased extracellular 
K+).  The uneven equilibration of concentration gradients results in 
changes in osmotic pressure with consequent flows of water.  Swelling of 
mitochondria and other structures occurs.  The appearance of "flocculent 
densities" in the mitochondria is thought to indicate severe internal 
membrane damage which is "irreversible."[ft. 16] 

Ischemic changes do not appear to result in any damage that would 
prevent repair (e.g., changes that would result in significant loss of 
information about structure) for at least a few hours.  Temporary and 
sometimes even permanent functional recovery has been demonstrated in 
optimal situations after as long as 60 minutes of total ischemia[93, 94, 
95].  Hossmann, for example, reported results on 143 cats subjected to 
one hour of normothermic global brain ischemia[97].  "Body temperature 
was maintained at 36 to 37 degrees C with a heating pad. ... Completeness of 
ischemia was tested by injecting 133 Xe into the innominate artery 
immediately before vascular occlusion and monitoring the absence of 
decay of radioactivity from the head during ischemia, using external 
scintillation detectors. ...  In 50% of the animals, even major 
spontaneous EEG activity returned after ischemia....  One cat survived 
for 1 yr after one hour of normothermic cerebrocirculatory arrest with 
no electrophysiologic deficit and with only minor neurologic and 
morphologic disturbances."  Functional recovery is a more stringent 
criterion than the more relaxed information theoretic criterion, which 
merely requires adequate structural preservation to allow inference 
about the pre-existing structure.  Reliable identification of the 
various cellular structures is possible hours (and sometimes even days) 
later.  Detailed descriptions of ischemia and its time course[72, page 
209 et sequitur] also clearly show that cooling substantially slows the 
rate of deterioration.  Thus, even moderate cooling "postmortem" slows 
deterioration significantly.

     Lysosomes

The theory that lysosomes ("suicide bags") rupture and release digestive 
enzymes into the cell that result in rapid deterioration of chemical 
structure appears to be incorrect.  More broadly, there is a body of 
work suggesting that structural deterioration does not take place 
rapidly.

Kalimo et. al.[74] said "It is noteworthy that after 120 min of complete 
blood deprivation we saw no evidence of membrane lysosomal breakdown, an 
observation which has also been reported in studies of in vitro lethal 
cell injury[omitted references], and in regional cerebral 
ischemia[omitted references]."

Hawkins et. al.[75] said "...lysosomes did not rupture for approximately 
4 hours and in fact did not release the fluorescent dye until after 
reaching the postmortem necrotic phase of injury. ... The original 
suicide bag mechanism of cell damage thus is apparently not operative in 
the systems studied.  Lysosomes appear to be relatively stable 
organelles...."

     Messenger RNA and Protein

Morrison and Griffin[76] said "We find that both rat and human 
cerebellar mRNAs are surprisingly stable under a variety of postmortem 
conditions and that biologically active, high-molecular-weight mRNAs can 
be isolated from postmortem tissue. ... A comparison of RNA recoveries 
from fresh rat cerebella and cerebella exposed to different postmortem 
treatments showed that 83% of the total cytoplasmic RNAs present 
immediately postmortem was recovered when rat cerebella were left at 
room temperature for 16 h [hours] postmortem and that 90% was recovered 
when the cerebella were left at 4 degrees C for this length of time .... In 
neither case was RNA recovery decreased by storing the cerebella in 
liquid nitrogen prior to analysis. ... Control studies on protein 
stability in postmortem rat cerebella show that the spectrum of abundant 
proteins is also unchanged after up to 16 h [hours] at room 
temperature...."

     17 Million Year Survival of DNA

The ability of DNA to survive for long periods was dramatically 
illustrated by its recovery and sequencing from a 17 to 20 million year 
old magnolia leaf[81].  "Sediments and fossils seem to have accumulated 
in an anoxic lake bottom environment; they have remained unoxidized and 
water-saturated to the present day."  "Most leaves are preserved as 
compression fossils, commonly retaining intact cellular tissue with 
considerable ultrastructural preservation, including cell walls, leaf 
phytoliths, and intracellular organelles, as well as many organic 
constituents such as flavonoids and steroids[omitted references].  There 
is little evidence of post-depositional (diagenetic) change in many of 
the leaf fossils."

     Cell Cultures taken after "Death"

Gilden et. al.[77] report that "...nearly two-thirds of all tissue 
acquired in less than six hours after death was succesfully grown, 
whereas only one-third of all tissue acquired more than six hours after 
death was successfully grown in tissue culture."  While it would be 
incorrect to conclude that widespread cellular survival occurred based 
on these findings, they do show that structural deterioration is 
insufficient to disrupt function in at least some cells.  This supports 
the idea that structural deterioration in many other cells should not be 
extensive.

     Information Loss and Ischemia

It is currently possible to initiate cryonic suspension immediately 
after legal death.  In favorable circumstances legal death can be 
declared upon cessation of heartbeat in an otherwise revivable 
terminally ill patient who wishes to die a natural death and has refused 
artificial means of prolonging the dieing process.  In such cases, the 
ischemic interval can be short (two or three minutes).  It is 
implausible that ischemic injury would cause information theoretic death 
in such a case.

As the ischemic interval lengthens the level of damage increases.  It is 
not clear exactly when information loss begins or when information 
theoretic death occurs.  Present evidence supports but does not prove 
the hypothesis that information theoretic death does not occur for at 
least a few hours following the onset of ischemia.  Quite possibly many 
hours of ischemia can be tolerated.  Freezing of tissue within that time 
frame followed by long term storage in liquid nitrogen should provide 
adequate preservation of structure to allow repair[ft. 17].


MEMORY

It is essential to ask whether the important structural elements 
underlying "behavioral plasticity" (human memory and human personality) 
are likely to be preserved by cryonic suspension.  Clearly, if human 
memory is stored in a physical form which is obliterated by freezing, 
then cryonic suspension won't work.  In this section we briefly consider 
a few major aspects of what is known about long term memory and whether 
known or probable mechanisms are likely to be preserved by freezing.

It appears likely that short term memory, which can be disrupted by 
trauma or a number of other processes, will not be preserved by cryonic 
suspension.  Consolidation of short term memory into long term memory is 
a process that takes several hours.  We will focus attention exclusively 
on long term memory, for this is far more stable.  While the retention 
of short term memory cannot be excluded (particularly if chemical 
preservation is used to provide rapid initial fixation), its greater 
fragility renders this significantly less likely.

To see the Mona Lisa or Niagara Falls changes us, as does seeing a 
favorite television show or reading a good book.  These changes are both 
figurative and literal, and it is the literal (or neuroscientific) 
changes that we are interested in:  what are the physical alterations 
that underlie memory?

Briefly, the available evidence supports the idea that memory and 
personality are stored in identifiable physical changes in the nerve 
cells, and that alterations in the synapses between nerve cells play a 
critical role.

Shepherd in "Neurobiology"[38, page 547] said:  "The concept that brain 
functions are mediated by cell assemblies and neuronal circuits has 
become widely accepted, as will be obvious to the reader of this book, 
and most neurobiologists believe that plastic changes at synapses are 
the underlying mechanisms of learning and memory."

Kupfermann in "Principles of Neural Science"[13, page 812] said: 
"Because of the enduring nature of memory, it seems reasonable to 
postulate that in some way the changes must be reflected in long-term 
alterations of the connections between neurons."

Squire in "Memory and Brain"[109, page 10] said:  "The most prevalent 
view has been that the specificity of stored information is determined 
by the location of synaptic changes in the nervous system and by the 
pattern of altered neuronal interactions that these changes produce.  
This idea is largely accepted at the present time, and will be explored 
further in this and succeeding chapters in the light of current 
evidence."

Lynch, in "Synapses, Circuits, and the Beginnings of Memory"[34, page 3] 
said:  "The question of which components of the neuron are responsible 
for storage is vital to attempts to develop generalized hypotheses about 
how the brain encodes and makes use of memory.  Since individual neurons 
receive and generate thousands of connections and hence participate in 
what must be a vast array of potential circuits, most theorists have 
postulated a central role for synaptic modifications in memory storage."

Turner and Greenough said "Two non-mutually exclusive possible 
mechanisms of brain information storage have remained the leading 
theories since their introduction by Ramon y Cajal [omitted reference] 
and Tanzi [omitted reference].  The first hypothesis is that new synapse 
formation, or selected synapse retention, yields altered brain circuitry 
which encodes new information.  The second is that altered synaptic 
efficacy brings about similar change."[22].

Greenough and Bailey in "The anatomy of a memory: convergence of results 
across a diversity of tests"[39] say:  "More recently it has become 
clear that the arrangement of synaptic connections in the mature nervous 
system can undergo striking changes even during normal functioning.  As 
the diversity of species and plastic processes subjected to 
morphological scrutiny has increased, convergence upon a set of 
structurally detectable phenomena has begun to emerge.  Although several 
aspects of synaptic structure appear to change with experience, the most 
consistent potential substrate for memory storage during behavioral 
modification is an alteration in the number and/or pattern of synaptic 
connections."

It seems likely, therefore, that human memory is encoded by detectable 
physical changes in cell structure and in particular in synaptic 
structure.

     Plastic Changes in Model Systems

What, exactly, might these changes be?  Very strong statements are 
possible in simple "model systems".  Bailey and Chen, for example, 
identified several specific changes in synaptic structure that encoded 
learned memories from sea slugs (Aplysia californica) by direct 
examination of the changed synapse with an electron microscope[36].

"Using horseradish peroxidase (HRP) to label the presynaptic terminals 
(varicosities) of sensory neurons and serial reconstruction to analyze 
synaptic contacts, we compared the fine structure of identified sensory 
neuron synapses in control and behaviorally modified animals.  Our 
results indicate that learning can modulate long-term synaptic 
effectiveness by altering the number, size, and vesical complement of 
synaptic active zones."

Examination by transmission electron microscopy in vacuum of sections 
100 nanometers (several hundred atomic diameters) thick recovers little 
or no chemical information.  Lateral resolution is at best a few 
nanometers (tens of  atomic diamters), and depth information (within the 
100 nanometer section) is entirely lost.  Specimen preparation included 
removal and desheathing of the abdominal ganglion which was then bathed 
in seawater for 30 minutes before impalement and intrasomatic pressure 
injection of HRP.  Two hours later the ganglia were fixed, 
histochemically processed, and embedded.  Following this treatment, 
Bailey and Chen concluded that "...clear structural changes accompany 
behavioral modification, and those changes can be detected at the level 
of identified synapses that are critically involved in learning."

The following observations about this work seem in order.  First, 
several different types of changes were present.  This provides 
redundant evidence of synaptic alteration.  Inability to detect one type 
of change, or obliteration of one specific type of change, would not be 
sufficient to prevent recovery of the "state" of the synapse.  Second, 
examination by electron microscopy is much cruder than the techniques 
considered here which literally propose to analyze every molecule in the 
structure.  Further alterations in synaptic chemistry will be detectable 
when the synapse is examined in more detail at the molecular level.  
Third, there is no reason to believe that freezing would obliterate the 
structure beyond recognition.

     Implications for Human Memory

Such satisfying evidence is at present confined to "model systems;" what 
can we conclude about more complex systems, e.g., humans?  Certainly, it 
seems safe to say that synaptic alterations are also used in the human 
memory system, that synaptic changes of various types take place when 
the synapse "remembers" something, that the changes involve alterations 
in at least many thousands of molecules and probably involve mechanisms 
similar to those used in lower organisms (evolution is notoriously 
conservative).

It seems likely that knowledge of the morphology and connectivity of 
nerve cells along with some specific knowledge of the biochemical state 
of the cells and synapses would be sufficient to determine memory and 
personality.  Perhaps, however, some fundamentally different mechanism 
is present in humans?  Even if this were to prove true, any such system 
would be sharply constrained by the available evidence.  It would have 
to persist over the lifetime of a human being, and thus would have to be 
quite stable.  It would have to tolerate the natural conditions 
encountered by humans and the experimental conditions to which primates 
have been subjected without loss of memory and personality (presuming 
that the primate brain is similar to the human brain).  And finally, it 
would almost certainly involve changes in tens of thousands of molecules 
to store each bit of information.  Functional studies of human long term 
memory suggest it has a capacity of only 10^9 bits (somewhat over 100 
megabytes)[37] (though this did not consider motor memory, e.g., the 
information storage required when learning to ride a bicycle).  Such a 
low memory capacity suggests that, independent of the specific 
mechanism, a great many molecules are required to remember each bit.  It 
even suggests that many synapses are used to store each bit (recall 
there are perhaps 10^15 synapses - which implies some 10^6 synapses per 
bit of information stored in long term memory).

Given that future technology will allow the molecule-by-molecule 
analysis of the structures that store memory, and given that such 
structures are large on the molecular scale (involving tens of thousands 
of molecules each) then it appears unlikely that such structures will 
survive the lifetime of the individual only to be obliterated beyond 
recognition by freezing.   Freezing is unlikely to cause information 
theoretic death.


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