NetNews Usenet Archive 1992 #27/NN_1992_27.iso/spool/sci/cryonics/652

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From: [e-mail] (Ralph Merkle)
Subject: The Technical Feasibility of Cryonics; Part #2
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The Technical Feasibility of Cryonics

PART 2 of 5.

by

Ralph C. Merkle
Xerox PARC
3333 Coyote Hill Road
Palo Alto, CA 94304
[e-mail]

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


----------------------------------------------------------

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.