X-Message-Number: 9762
From: Ralph Merkle <>
Subject: Repost from January 1993
Date: Sat, 23 May 1998 10:55:48 PDT

The following repost is from January of 1993:


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Comments On "The Effects of Cryopreservation on the Cat"

Mike Darwin recently posted "The Effects of Cryopreservation on the
Cat" by Michael Darwin, Jerry Leaf, and Hugh Hixon. This is "...a
research paper which is now in the (hopefully) final stages of
preparation for publication."

Before discussing the paper by Darwin et. al., we need to discuss
the general objectives of research in cryonics.

Fundamentally, cryonics aims to prevent the terminally ill patient
from dying when today's medical technology can't.  We stabilize the
patient's condition as best we can using today's technology, and
then maintain the patient in a stable condition until future 
medical technology can revive them.  The method used is to store the 
patient in liquid nitrogen.

It is generally agreed that the condition of a person stored in 
liquid nitrogen is stable.  However, the process of freezing 
inflicts damage.  The question of interest, therefore, is whether 
future medical technology can revive the patient despite the damage 
inflicted by the freezing process, and despite any other disease or 
injury from which the patient might have suffered.

The first question is whether any future technology could revive 
the frozen patient, even in principle.  If revival is infeasible in 
principle, then the resources allocated for cryonic suspension 
might be better used for other activities (Bacchanalian orgies come 
to mind).  This line of thought leads to the information theoretic 
criterion of death (discussed more extensively in "The Technical 
Feasibility of Cryonics," by Ralph C. Merkle, Medical Hypotheses, 
September 1992, 39, pages 6-16; and elsewhere).  If information 
theoretic death has occurred then cryonics won't work.  The hopes, 
dreams, memories, and personality of the person have been 
obliterated in the information theoretic sense and no future 
technology, regardless of how advanced, can recover them.  They are 
gone.

If, on the other hand, information theoretic death has not occurred 
then revival is feasible in principle.  In this case, we must ask 
whether revival will prove feasible in practice given that it is 
feasible in principle.  Until relatively recently most people would 
have said there's a big difference between "in principle" and "in 
practice."  Research in molecular manufacturing, however, tells us 
that repair "in principle" and repair "in practice" will, at some 
point in the future, likely become almost indistinguishable.

This point deserves further emphasis.  The condition of a person 
frozen at the temperature of liquid nitrogen is quite stable.  The 
recovery of the X, Y and Z coordinates of every atom in the frozen 
structure should in principle be feasible.  With the advent of the 
STM and other scanning probe microscope techniques, it has become 
clear that the recovery of such detailed structural information 
(e.g., the coordinates of every atom) is possible.  
Molecular manufacturing should let us economically build large 
numbers of very small STM-like devices able to analyze frozen 
tissue in complete detail.  Thus, by combining low-cost 
manufacturing with high-resolution imaging systems, we can recover 
essentially all the information about the structure that can in 
principle be recovered.  By combining such analytical tools with 
systems able to change molecular structure in a general way, we 
will then have (metaphorically speaking) eyes with which to see and 
hands with which to heal.  Even damage at the molecular level will 
then be susceptible to repair.  As Feynman put it, "The problems of 
chemistry and biology can be greatly helped if our ability to see 
what we are doing, and to do things on an atomic level, is 
ultimately developed -- a development which I think cannot be 
avoided."

Thus, the fundamental objective of today's cryonic suspensions is 
to maximize the likelihood of information theoretic survival.  One 
of the major purposes of research in cryonics is to determine if 
current suspension methods prevent information theoretic death.
Before considering the other purposes of research in cryonics, it 
is useful to ask what conclusions (if any) can be reached about 
information theoretic survival based on the paper by Darwin et. al.

At the outset, I should say that I have not yet had an opportunity 
to examine the pictures that the paper is based on, and so must base 
my comments on the text and conversations with Mike.  I have, 
however, examined some of the pictures produced by Fahy of rabbit 
brain (which are also relevant in the current discussion).  I 
should also say quite clearly that I greatly appreciate the work 
and effort that went into this study, and feel that it makes a 
valuable contribution to our understanding of the effects of 
cryopreservation. The comparison that follows, illustrating the 
great power of future technologies in comparison with current 
technologies, should in no way be construed to mean that we should 
not vigorously pursue research on the effects of cryopreservation. 
It is intended as a caution against overinterpreting the 
experimental data that we obtain with today's admittedly imperfect 
methods.

We are faced with a fundamental dilemma in trying to determine with 
today's technology whether future technologies will decide that 
information theoretic death has (or has not) taken place.  In the 
future, we will have complete information about every molecule in 
the frozen structure.  In the present study, we have information 
provided by light and transmission electron microscopy.  Further, 
the information is about the structure after it has been thawed, 
fixed, and sectioned.

Even under the best of conditions, the information available using 
transmission electron microscopy is grossly poorer than the 
information that will be available in the future.  We learn only the 
electron density of the imaged section.  If the section is 1 micron
thick (plausible, although the actual section thickness used in
the study was not specified) then resolution of detail much smaller
than 0.1 microns (0.1 of the section thickness) is difficult.
We will assume, however, that a resolution of one hundredth of the 
section thickness, 0.01 microns or 10 nanometers, was in fact 
achieved.  Under these conditions a single pixel of our EM 
photograph will be available for every rectangular block that is 
one micron (or 1,000 nanometers) long by 10 nanometers by 10 
nanometers.  Assuming we are getting 10 bit grey scale data (again 
optimistic), this means we have 10 bits of information for a volume 
of 100,000 cubic nanometers.  There are (very roughly) 100 atoms 
per cubic nanometer, so we have 10 bits for 10,000,000 atoms, or 1 
bit for 1,000,000 atoms.  While we might reasonably debate the 
number of bits of information that future systems will generate, it 
is reasonable to suppose that they will give us at least 1 bit per 
atom of raw information (again conservative), in the same way that 
EM photography gives us 10 bits per pixel of raw data.  We thus have 
at least a factor of 1,000,000 less information about the frozen 
structure when we are looking at an EM photograph than will be 
available in the future.  To put this a bit more graphically, if you 
are looking at a computer screen with 1,000 x 1,000 resolution 
(perhaps a 21" monitor) then our factor of 1,000,000 less 
information is the same as taking a complex scene portrayed on this 
21" screen and crushing it into a single dot with an equivalent 
"screen size" of a 50th of an inch.  The ability to discern 
biologically significant structure despite such rudimentary imaging 
methods is quite remarkable and speaks volumes about the great 
redundancy in such structures.

The difference is actually more dramatic when we consider that, 
qualitatively, an EM section gives us no depth information (unless 
we use serial sections, a method not employed in the current 
study).  Thus, we have only a fuzzy view of the projection of a 
single slab taken with a random orientation through a complex three 
dimensional structure after it has been subjected to warming, 
fixing, deglycerolization, and the mechanical insults of 
sectioning.

While the paper did a good job of pointing out the inherent 
weaknesses in the methods used, it should also be pointed out that 
the method of fixation used for the control group (vascular 
perfusion in situ) was different from the method used to fix the 
experimental group (removal en bloc followed by immersion in a 
fixative-containing solution).  Vascular perfusion in situ is 
superior and offers fewer opportunities to introduce artifacts.  To 
quote Palay et. al. from "Fixation of Neural Tissues for Electron 
Microscopy by Perfusion with Solutions of Osmium Tetroxide:"  "The 
difference between the devastated tissue resulting from immersion 
fixation and the coherent, intact tissue obtained by perfusion 
fixation is at once evident on even superficial examination."
Thus, the introduction of artifacts because of this difference in 
protocol between the experimental and the control group cannot be 
excluded.

It should also be remembered that the 3 molar glycerol used is less 
than the "Smith's Criterion," and is substantially less than the 5 
to 6 molar glycerol used in today's suspensions.  The ischemic cat 
("FIGP" in the terminology of the paper) was subjected to a 24 hour 
ischemic interval.  Under good conditions today (certainly not 
always achieved, but achieved sometimes) the ischemic interval can 
be held to a few minutes.  In the case of the terminally ill patient 
who has elected to forego artificial methods of prolonging the 
dieing process, suspension can quite literally be started shortly 
after cessation of heartbeat at a point in time when the patient 
could in fact be revived by current methods.

It is also clear that freezing to liquid nitrogen temperatures 
introduces macroscopic fractures as the temperature is reduced 
below the glass transition temperature (at about 130 Kelvins) to 
the 77 Kelvins of liquid nitrogen.  The paper also suggests that
smaller fractures exist.  Fractures created at or below the glass 
transistion temperature result in little or no loss of significant 
structural information.  From an information theoretic point of 
view, provided that the tissue remains frozen both prior to and 
during analysis, the presence of fractures is not a major concern 
and is unlikely to cause information theoretic death.  Upon 
rewarming, however, such fractures will clearly contribute to 
artifacts and result in loss of cellular contents and structure.
Again, the paper correctly points out this mechanism for the 
introduction of artifacts, and proposes further studies which 
freeze to the glass transition temperature and not below to 
eliminate this source of error.

It would also be most advisable for a future revision of the paper 
to state clearly that future analysis on the frozen structure is 
anticipated.  Attempting to infer the correct structure after 
thawing significantly overstates the problem.

The paper clearly establishes that gross macroscopic fractures are 
present.  As mentioned earlier, fractures that occur at or below 
the glass transition temperature are unlikely to cause significant 
loss of structural information (though they are likely to preclude 
revival by any technology short of a medical technology that fully 
utilizes a mature nanotechnology).  Unless one is eager to be 
revived using primitive or intermediate technologies (which might 
be able to cure most, though not all, injuries) the presence of 
fractures is of little direct concern.  Two secondary concerns are 
(a) the greater deterioration that would occur if a (hopefully 
unlikely) thaw-refreeze event were to occur and (b) the negative 
appeal of fracture damage to many people.  Should storage at the 
glass transition temperature become available it would be 
marginally preferable, but only if it were certain that it was as 
reliable as the current highly reliable method (e.g., pouring 
liquid nitrogen into a large dewar once every few weeks).

Blockage of the circulatory system in the ischemic group is of 
serious concern.  Long ischemic intervals are sometimes 
unavoidable in current suspensions, particularly given the poor 
social and legal environment.  While blockages in the central 
nervous system created "grossly visible infarcted areas..."
"...these were relatively few" and generally "no larger than 2 mm 
to 3 mm in diameter..."  This essentially means that the areas which 
did not receive cryoprotection were subjected to a straight freeze.
While I would suspect that survival following a straight freeze is 
likely, there is insufficient data at the present time to support 
such a conclusion with confidence.

In the non-ischemic group, dehydration increased the difficulty of 
identifying structures.  As discussed previously, future methods 
should have no difficulty in identifying structures that are 
obscure today.  Irregularly shaped cavities were present, 
presumably formed during freezing by the growth of blocks of ice.
The slow growth of ice during freezing is likely to cause 
compression of tissue.  Compression is of little concern from an 
information theoretic point of view.  More significant damage 
(e.g., tears, rips, or microfractures) were also present.  Given 
the evidence in other systems that substantial ice formation is 
compatible with functional recovery, it is likely that either (a) 
the observed damage is compatible with functional recovery, or (b) 
it occurred after most of the water had frozen (and presumably 
after most of the damage caused by freezing had occurred).  In 
either case, information theoretic loss should not be great.

The presence of tears or rips are also seen in the freeze 
substituted preparations of rabbit brain prepared by Fahy.  In that 
case, the tears appeared to be "clean" with matching surfaces, 
making hypothesis (b) more likely.

The presence of unidentified "organized debris" in the spaces 
presumably created by ice during the freezing process might have 
occurred either during freezing or thawing.  The hypothesis that 
the debris was moved to the space during freezing is complicated by 
the observation that the space was, at that time, occupied by ice.
After thawing, the volume occupied by ice would become a small pool 
of water.  Anything which broke free from the wall or lining of such 
a pool would then drift freely in the pool, thus creating debris.
An attractive and simple hypthesis for the formation of the debris 
during thawing is available, while hypotheses for the formation
of debris during freezing face significant difficulties.

The increasingly dehydrated and confined regions between the blocks 
of ice formed during freezing should make movement of any structure 
of significant size quite difficult.  While there might be some 
concern that the currents created during freezing will result in 
turbulent flow, this appears quite unlikely.  [The following
discussion has been revised and corrected from the original post] The
approximate  criterion for the onset of turbulence in a liquid volume
with  characteristic size r is that the Reynolds number ~rdv/n exceed 
a few thousand, where d is the density of the liquid, v the velocity of 
the flow, and n the viscosity.  The characteristic dimensions in a 
cell are about a micron, the density is roughly a gram per cubic 
centimeter (or 1,000 kilograms/cubic meter), the velocity is
probably much less than a meter per second (and probably much less
than a micron per second), and the viscosity of water at room
temperature is about 0.01 poise (or 0.001 newton-second/meter^2)
(viscosity increases both with decreasing temperature and with an
increasing concentration of glycerol, so 0.01 is conservative).  This 
produces a Reynolds number less than 1.

As the approximate criterion for the onset of turbulent flow is a
Reynolds number of a few thousand or more, this implies that
turbulent flow is implausible.  When we take into account the fact
that the viscosity of tissue is larger than the viscosity of water
(assuming that we can apply "viscosity," in an approximate way,
to a structure as heterogenous as tissue), that the tissue is
loaded with glycerol (which has a higher viscosity than water),
that the whole system has been chilled to some low temperature
(which also increases viscosity) and that the velocity of flow will
typically be substantially less than a meter per second, then we
conclude that the actual Reynolds number will be significantly
smaller than 1.  This reinforces the basic conclusion.

The "Encyclopedia of Physics" says "Blood flow in capillaries is laminar,
but water flow in household pipes is turbulent unless the flow is about
that allowed by a leaky faucet or less."

Another concern is that two small pieces of tissue, originally 
adjacent, might be subjected to differential forces that would 
cause one to move past the other for some distance.  The examples 
that have on occasion been used by Mike Darwin are boulders moved by 
glaciers or bits of debris moved by the formation of ice on window 
panes.

In both these cases, water is converted almost completely into ice.
In the case of a cryonic suspension in the presence of even moderate 
amounts of cryoprotectant, freezing is incomplete and the result is 
more similar to slush than ice.  In addition, in both the examples 
cited there was a rigid surface which provided an attachment point 
for the ice.  The idea that slush might create a differential force 
on two adjacent points of tissue otherwise suspended in solution is 
more difficult to envision.

The damage to the axons of myelinated nerve cells, secondary to a 
failure of the cryprotectant to penetrate the myelination (many 
layers of cell membranes wrapped tightly around the axon) is very 
plausible.  The function of a myelinated axon, however, is to carry 
information (much like a wire).  Complete obliteration of the axon, 
analagous to damage to a wire, will result in little or no 
information loss if the myelin sheath (somewhat like the insulation 
around a wire) is still present.  Myelinated axons are relatively 
large, so even substantial damage to the axon would not obscure or 
obliterate the pathway.

Turning to the ischemic group, we find that axons in myelinated 
tissue were sometimes sufficiently damaged that "nothing but debris 
remained."  Even if the axonal debris were to prove unidentifiable, 
the course of the neuron would still not be obscured.  As discussed 
earlier, however, the application of future analytical tools will 
almost certainly resolve the nature of "unidentified" EM images.

In summary: the available evidence, though clearly incomplete, tends
to support the idea that information theoretic survival is likely even
when today's rather primitive suspension methods are used.  This should 
not be taken as a reason for discontinuing or ignoring research in 
this area.  Even a moderate risk of dying is unacceptable and 
should be reduced.

A more pressing motivation for research has little if anything to 
do with information theoretic survival per se.  The most serious 
risks to survival stem from the more or less complete failure of the 
medical community to either understand or support cryonics.  This 
failure leads directly to preventable delays in initiating 
suspensions, inadequate support for suspensions if they are 
tolerated, etc.  The single most effective method of decreasing the 
risk of death would be to gain even a moderate level of acceptance 
from the mainstream medical community.  To gain such acceptance 
will require a body of research which supports the idea that 
suspension protocols do in fact provide a good chance of survival.
For various reasons it seems likely that such research will have to 
provide almost conclusive evidence that cryonics is likely to work, 
despite the obvious disadvantages of requiring "proof" that 
cryonics works before using it.  The idea that freezing a person is 
a "risky" course of action while cremation and burial alive are 
"conservative" is quite absurd, but also deeply entrenched.

Thus, along with information theoretic survival, the second main 
objective of research is the fuzzier one of gaining general 
acceptance by the medical community.  Therefore, besides 
understanding current suspension protocols and improving future 
protocols measured against the criterion of information theoretic 
survival, we must also understand and improve suspension protocols 
measured against the (somewhat fuzzy) criterion used by the 
mainstream medical community.

While it is more difficult to specify exactly what will be needed to 
satisfy this second criterion, the two obvious objectives are (1) 
demonstrate reversible cryopreservation of a mammalian brain, e.g., 
freeze and thaw the brain of an animal and show functional recovery
for at least a short period of time following thawing; or
(2) demonstrate that suspension techniques, while they do not
preserve function, provide good preservation of the structures that
are crucial to the correct functioning of the human brain and memory,
e.g., get pictures (either from light or electron microscopy) that
show good preservation of structure and ultrastructure.

There is much work to be done to develop this body of research, and
all efforts in this direction should be encouraged.

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