X-Message-Number: 15505
Date: Wed, 31 Jan 2001 13:20:15 -0500
From: Paul Antonik Wakfer <>
Subject: Ongoing Cryoscience Comments - CryoNet #15482, 15483, 15485, 15489, 
References: <>

> Message #15482
> From: "Jeff Grimes" <>
> Subject: Mr. Ettinger's Ideas about Medicine
> Date: Tue, 30 Jan 2001 16:26:38 +0000
> Mr. Ettinger writes:


> > In cryonics procedures also, the bottom line is results. The results of CI
> > experiments have been evaluated by independent professionals--two sets of
> > them--and key portions of the reports are on our web site.
> Well, wait a minute. One of your independent professionals was the biologist
> who did the original sheep head research, described on your web site, is
> that right?

Frankly, I don't know what kind of "professional" biologist would
attempt to experiment with sheep heads obtained from a slaughterhouse!
Since no exact control and prescription of the handling of experimental
subjects from life to end processing is possible, no true science is
possible either. You will find damned few biology experiments in 
peer-reviewed publications which involves analysis of life, which begin
with dead animals, and most certainly none which begin with animals
procured from slaughterhouses. This is because the full procedure and
timing of all handling from live animal to final processing is critical
in order to get repeatable, and therefore meaningful results. For
example, expect for pathology work, which is not for similar purposes to
what we are discussing here, medical research never does experiments on
cadavers, the results of which are meant to apply to live persons.

> But your web site suggests he was working for you. So how
> independent can he be? The other professionals would be the people in Canada
> who did the more recent study. Is that right? But you won't even tell us who
> they are!

And what have they done in the past which is relevant to their ability
to critically and valuably analyze the CI research?

> Message #15483
> From: "Jeff Grimes" <>
> Subject: Procedures that don't seem to make sense
> Date: Tue, 30 Jan 2001 16:30:29 +0000
> Reading back through the Cryonet posts I missed last week, I am surprised
>(and somewhat confused) to find Douglas Skrecky stating that the 75% solution
> of glycerol used by Cryonics Institute is actually concentrated enough to
> vitrify! It is a vitrifying solution. So, Mr. Ettinger has denounced
> vitrification as destructive, toxic, and unnecessary, but his organization
> is in fact using a solution of glycerol that is strong enough to induce
> vitrification?

I think this is a much higher concentration than was being used by all
cryonics organizations before 1992. It was one of the toxic
concentrations which were known to vitrify but were discarded. However,
"known to vitrify" must be explained here.
While 75% may vitrify if cooled all by itself, in order to vitrify a
piece of tissue it must reach that same uniform concentration throughout
the whole piece of tissue (this is the meaning of equilibrate). However,
glycerol is so viscous and impermeable at a 75% concentration, that this
equilibration must either be done at a high temperature (where viscosity
is reduced) or for a very long time at a lower temperature.
Unfortunately, since the toxic damage done by a chemical is at least
proportional to both the temperature and the time, neither of these
options will reduce the toxicity to acceptable levels. That is why the
original decision was made that it was better to have some ice damage
than to have the toxicity of highly concentrated glycerol or any other
cryoprotectant which was known to vitrify before 1992. I suspect that CI
procedures were changed sometime in the 90s, perhaps in response to
Alcor's glowing (but wrong, IMO) reports of proudly reaching very high
glycerol concentrations with their patients.

Since Mr Ettinger has admitted elsewhere that the one pass 75% glycerol
procedure does not allow tissue to equilibrate (nor will his proposed
4-step procedure, BTW), this implies that near the vasculature the cells
are suffering toxic damage, a little further away they are probably not
getting to high enough concentrations to suffer much damage but they are
not vitrifying either although the ice damage may be tolerable, and the
most remote tissue portions may be getting close to a straight freeze
and suffering major ice damage. I must say that I really don't know what
to make of this. Is it the purpose of CI to attempt to cryopreserve its
patients in all possible ways at the same time, in order to hedge its
bets and be sure that some tissue comes back whatever turns out to be
best and whatever the future holds?

Finally, we now have an explanation for why CI never sees any
macroscopic cracking of its patients!
With this kind of non-uniform material it is much more likely for the
stresses engendered by lowering the temperature to that of liquid
nitrogen (more than necessary as has always been known, but a
convenience for inexpensive storage) can be relieved by non-cracking
distortions of the tissue.
> When Mr. E wrote on his web site that "the cryoprotectants used in
> vitrification are toxic ... to the point of actually disintegrating
> cell membranes" did he realize that a) the solution used by his own
> organization is strong enough to vitrify, and b) his stuff is actually
> MORE toxic than the new vitrification solutions being developed?

At this point, I need to make a retraction of a previous statement which
I made some time ago on CryoNet.
In conversation, with one of the scientists at 21CM I have been
convinced that while highly concentrated glycerol is highly toxic, there
is no solid evidence that it disintegrated cell membranes. This is
certainly true for some other cryoprotectants at vitrification
concentrations, but not for glycerol.
> Also, since the open-ended perfusion process may produce such uneven
> effects, I wonder if tests at the lab in Canada produced a big range
> of results, from bad to good, depending which piece of tissue was under
> the microscope. I wonder if the CI web site shows this full range, or
> includes only the better results.

Actually, if this is so then CI is not alone in this kind of selective
reporting, for it is a major problem in establishment science research
also. It is the major reason why experiments are technically done
without a "purpose", but only to discern what "happens" when certain
procedures are performed. However, too often "negative" results are not
published and do not receive additional funding. Unfortunately, this
biases the overall results of science. An example of this is a fully
performed and publishable lifespan experiment on mice using CoQ10 which
Steve Harris performed many years ago while working with Roy Walford.
Even though the CoQ10 beautifully "squared" the mortality curve, the
"purpose" of the experiment was not obtained since the maximum lifespan
of the mice was not increased. This experiment was never published and
its results remain outside the boundaries of scientific evidence.

> One last point. David Pascal said that the "open ended" perfusion used
> at CI is better because it takes less time, and consequently the person
> doesn't suffer so much deterioration at relatively warm temperatures.

If a good transport blood substitute were placed into the patient first,
as is done for organs to be transplanted, then little deterioration
would take place at a temperature just above 32' F. This was proved by
many dog experiments performed first by Mike Darwin and Jerry Leaf in
the mid 1980s and later in 1992-95 by Mike Darwin at Cryovita
Laboratories and 21CM.

> Also, when the perfusion is over, the patient is cooled VERY slowly,
> at less than 1 degree per hour. At Alcor, I am told that the rate is
> 10 degrees per hour, to get the temperature down as quickly as possible
> to minimize deterioration, or chemical damage from cryoprotectant. (All
> chemical reactions apparently occur more slowly at lower temperatures.)

It looks like CI has greatly raised their glycerol concentration without
realizing that this requires a consequent adjustment of the cooling rate
from what it (correctly) was with a lower concentration.

> Message #15485
> From: 
> Date: Tue, 30 Jan 2001 12:06:25 EST
> Subject: more Grimes


> 4. Does lack of equilibration mean that glycerol is unevenly distributed in
> the tissues of our experimental animals and patients? That is a reasonable
> suspicion, but actual findings (specimens from different sites in the brain)
> do not indicate that.

This is very strange to say the least. Having been involved with many
cryopreservations, mixed the perfusate (glycerol) for several, and also
seen it perfused into several experimental animals (not to mention from
theoretical grounds alone based on permeability calculations), it is
extremely hard to get high concentration glycerol to equilibrate
(distribute uniformly throughout the tissue). In fact, as I have said
before, this can only be done if the perfusion takes place at a very
high temperature (far above 10'C at which we perfused patients at much
lower concentrations) or if the perfusion is closed circuit and takes an
inordinate amount of time. For 75% glycerol to equilibrate in one pass
is simply impossible! Thus, these "actual" findings" are now totally
suspect, IMO.

> In any case, we have now changed over to a 4-stage stepped glycerol
> procedure, which will be outlined on our web site soon, replacing our current
> segment on Phases of Suspension.

This will also not likely allow equilibration at the 75% concentration.
The only way equilibration is certain to be reached is by using a
recirculating system. One starts with a low concentration and circulates
it for a while until its measured concentration is starting to decrease
(it does this because as the glycerol enters the tissue, water is pulled
out of the tissue and into the circulating perfusate). Then one removes
some of the lowered concentration perfusate and adds some of the very
high concentration. This process is now iterated until such time as the
input and output concentration are equal to each other and equal to the
desired tissue concentration and have been at these values for some
time. I don't remember the exact numbers used for timing, but this is
the general approach.
> >R.E. wrote: "As for Grimes, however--Alcor is welcome to him."
> >Why on earth would CI try to get rid of someone who simply wants to know how
> >things work?
> I have not said we would turn him down, but it ought to be obvious why I
> would not be eager to sign up someone like this. Look at the history of other
> organizations. Alcor had endless internal bickering, because of the
> personalities of some of its people, and there was a major schism resulting
> in the formation of another organization, CryoCare. Trans Time and the Bay
> Area Cryonics Society (now American Cryonics Society) had serious squabbles,
> and the initial cooperation between BACS and TT broke down. There were
> serious squabbles involving CryoSpan and related organizations or companies,
> and CryoSpan appears about to fold.

First, their is a major difference between situations where people
cannot work together amicably because of major personal differences in
life views and concerns, and where they differ dramatically in their
of which methods are most scientifically supportable. I don't know about
the ACS/TT feud (I never have understood it), but the CryoCare/Alcor
split was mostly not about science and cryopreservation methods (except
perhaps about similar methods being executed badly). Also, there never
were any squabbles between CryoSpan and anyone else, any dissension was
purely between yours truly and some others (mostly Mike Darwin).
However, even there, both of us were and remain admirers of each other's
scientific knowledge and methods. Finally, CryoSpan is not folding
because of any ongoing "squabbles" but because those in charge of it
wish to spend more of their scarce time on cryopreservation research
which is where our future lies. All CryoSpan patients will be
to other organizations as some have been already, before it "folds".
These sorts of differences are bound to happen in a "fringe" area such
as cryonics which by its nature attracts independent minds. IMO, it is
far better to have this than a bunch of unquestioning yes-men who have
little indepth understanding gathered around the revered founder.

> The fact that CI has had a much more stable history does NOT mean that our
> directors rubber-stamp my opinions. Our directors think for themselves, and I
> have been overruled more than once, in fact frequently. But our people
> generally do not have inflated egos or sand-paper personalities, and we don't
> need that.

If you are referring to Mr Grimes, I haven't seen a single thing which
suggests an "inflated ego".

> 7. In #15474 he asks:
> > Do other organizations use the glycerol before the person is moved to the
> cryo lab, or is this a feature which is unique to CI?
> Yes, CPA perfusion by funeral directors is unique to CI. Except in rare
> cases, it must be arranged well ahead of time.

Presumably this also means that no measurements of glycerol effluent
concentration were made (or even verification of the perfusate mixture
concentration) by means of a refractometer which is a rather expensive
instrument for each mortician to have just for this purpose.
In addition, how stable are CI perfusates? How long are they stored with
morticians before replacement? All medical perfusates have
scientifically necessary expiration dates based on stability. The
which BPI used (and I often mixed) was not stable and had to be mixed
fresh every time.

> 8. He writes:
> > After you wash out the blood, it would be easy enough to use a standard
> organ >cryopreservation solution,
> There isn't any "standard organ cryopreservation solution"--organs for
> transplant are not cryopreserved.

I expect Mr Grimes, being a novice, erred here and meant to say
transport blood substitute solution of which there are several standard
ones, UW solution being a favorite.

> Message #15489
> From: 
> Date: Tue, 30 Jan 2001 16:27:17 EST
> Subject: Wakfer
> Wakfer's latest (#15478) is tiresome, pretentious and offensive
> pontificating,

Perhaps Mr Ettinger equates an attempt to educate and to precisely and
logically explain complex concepts with pontificating. If so, I have no
idea from where he got that idea. It is, of course, an old dodge to
denigrate ones critic by impugning his character. If Mr Ettinger is
tired ("tiresome") perhaps one of his well-known afternoon naps will
help renew him for the challenge of truth and exact use of language.
> but I will point out a couple of things. First, consider this
> Wakfer gem:
> >There are no accepted views of what preservation "methods" are best for
> >brains, since "best" implies a specific purpose, and the purposes of
> >some uses of brains are not at all the same as other uses. Finally,
> >"best" is by its very nature not an objective of science which is the
> >investigation of reality and the discovery of the facts thereof. "Best"
> >is a purely subjective term of valuation and as such is totally
> >non-scientific. However, even in subjective practical terms, no one in
> >cryobiology or any other branch of science (except perhaps those at INC
> >and 21CM) currently has the purpose of restoring life as their purpose
> >for preserving brains!
> All clear now? On the one hand, for example, anyone who tries to find the
> "best" way to treat a disease is just not scientific. On the other hand, INC
> and 21CM do have a clear criterion for the "best" procedure in
> cryopreservation and are very scientific.

Once again Mr Ettinger has missed the point and/or purposefully
attempted to distort what I wrote by omitting his own preceding

>>>Would you compare that information with reports in the cryobiological 
>>>literature and make a theoretical guess as to whether our methods conform
>>>to "accepted" views of what is best for brains?

This paragraph makes it clear that "best" was referring to general
cryobiological practice with brains (actually there isn't any such
practice with whole brains), which does *not* have the purpose of
preserving brains to be restored to life! In that context my own
succeeding paragraph was completely valid and his criticism is out of
place. But let me nevertheless analyze his words.

> On the one hand, for example, anyone who tries to find the
> "best" way to treat a disease is just not scientific.

First, there is never only one "best" way to treat a disease. There are
usually many ways, some of which are best for some patients under
conditions and some of which are best for others under other conditions,
with "best" here being also variable depending on the values of the
patient and the treating physician. "Best" could means short term
quality of life, average patient mortality rate, percentage of total
patient recoveries, maximum after treatment patient lifespan, or any
number of other measured parameters.
Second, while the research about a disease may be scientific,
technically the treatment of patients with a disease is not, and
certainly the valuation of what is the "best" way to treat a disease is
highly subjective and not science at all.

> On the other hand, INC
> and 21CM do have a clear criterion for the "best" procedure in
> cryopreservation and are very scientific.

I never said this and it is not true. I cannot speak for 21CM, but INC
makes no statements whatsoever about what is "best" for
cryopreservation, or even "best" for anything at all. What we have
reported, and will publish at the appropriate time, are the measured
results of experiments executed according to the best currently accepted
scientific procedures. *That* is the end of science for this work. Any
interpretation or use of those reports for cryopreservation is separate
from science and is in fact mere speculation.

> Then there is a torrent of drivel,

Another wonderful phrase to denigrate an opponent who hits too close to

> the net import of which is that
> documentation of various possible measurements during a suspension procedure
> is more important than evaluation of the actual result.

Mr Ettinger has built a straw man to bash here. I said nothing about
*suspensions*. It was scientific experiments to which I was referring.
With every session of debate, I am finding it harder to believe that
these "missings of the point" are accidental or innocent errors of
oversight, rather than purposeful, conniving obfuscations and

> >intermediate dynamic measurements often tell much more than a few electron
> >micrographs (which after all are of an insignificant amount of the total
> tissue which was processed).
> Anyone who believes this is welcome to it.

To calculate the obvious from very rough estimates since I am familiar
with the generalities but not the exact numbers involved: Tissue samples
for electron micrographs are thin slices (perhaps a 1/10th of a
millimeter thick) of at most few millimeters on each side. The field of
view for detailed cellular examination is much smaller than this. Since
the size of a brain is of the order of hundreds of millimeters in each
of three dimensions (recall that 25.4 millimeters equals 1 inch), it is
very clear that EM appraisal can only "cover" an insignificant amount of
a whole brain unless an inordinate amount of work is done. This 
weakness is usually accounted for by the judicious use of representative
samples. However, if a large block of tissue is not equilibrated with
respect to all experimental parameters, it is not really possible to
find or choose "representative" samples.
> >>Two sets of independent professionals have evaluated our work,
> >Please state their professional qualifications.
> >Ie. what have they done which directly relates to what CI had them do?
> They are university faculty with specialties in electron microscopy,
> physiology, and pathology.

Thank you. It would be nice to know what university (there is a huge
variation) and to see a list of publications for each, but I will accept
that they have some expertise in some of the relevant areas. However,
neurophysiology and pathology at this delicate level is in itself a
specialized field. Do they have major direct experience with examination
of living brain tissue? 
> >from which portions of the tissue were the micrographs taken? Were these
> from >the highly glycerolized portions near the vasculature, the intermediate
> >concentration portions further away, or the nearly untouched portions
> farthest >from any glycerol perfusate?
> All of the above, with little difference.

If there was little difference then I submit that is prima face
evidence that the "professionals" did not know what they were doing and
did in fact not do a proper evaluation. Either that or such visual
examinations, even at the EM level are of very little value in
determining the "health" or "viability" of brain cells and tissue, OR
the tissue was all so bad that it all looked the same.
> >We do not know what is best and we *will not* know what is best until an
> animal >brain is restored with full functionality in all reasonably
> measurable parameters.
> Unbelievable! "Best" does not mean perfect, it means the procedure which,
> among those we have tried, has shown the best results, the least damage, by
> all the criteria we are able to apply.

An "Unbelievable!" back to Mr Ettinger. Without any restorations having
been done, it is logically impossible to state which current preparation
will allow the easiest, most complete, or any recovery at all. There is
no possible way to know what is the best procedure at the present time.
We will only know that at some time in the future. "All the criteria we
are able to apply" may in the future be found to be so much nonsense!
That having been clearly understood (which Mr Ettinger appears to be
incapable of doing), we can then proceed to apply the most reasonable
criteria that we can imagine, and we hope will be important for future
success in patient restoration.
> >If one doesn't know enough to evaluate one's own results, then one does
> >not know enough to be doing the experiment! To separate the evaluation
> >of results from the conduct of the experiment which produced them is not
> >feasible scientifically.
> Perhaps a new high for both pretentiousness and drivel. We are able to
> conduct many experiments. We are not equipped to do electron microscopy. We
> hire experts for this. I somehow doubt that most readers, "scientific" or
> not, will fault us for this.

I never said that you needed to have your own electron microscope.
However, you do need to be thoroughly familiar with the various methods
of specimen preparation, to select the precise areas of tissue from
which the specimens are taken, to specify the methods of specimen
preparation and other EM parameters which I know little about, and
finally to become thoroughly familiar with what you are looking at and
especially as it relates to other cryopreserved tissue samples from
past experiments of others. If you do not do this, then you are not
and adequately performing the experiment. This is how good science is
done. Nothing less will suffice. Anything less is not complete and can
be quite meaningless.

>Message #15493
>Date: Tue, 30 Jan 2001 23:02:21 EST
>Subject: rabbit note
>The paper is called "Bioelectric activity of cryopreserved brain pieces of a 
>rabbit," by Yu. I. Pichugin, V.S.Marchenko, and A.V. Shilo. First publication 
>was in The Immortalist in two parts, issues of September and November, 1995. 
>Other versions were at the 14th Conference on Preservation of Genetic 
>Resources, Pushchino, 1996; and the 5th European Conference on Tissue Banks, 
>Berlin, 1996.

Two things here.

1. Is the paper published in full form in the above conference
proceedings or only in abstract form?

2. It is a well established principle in science that a paper published
even in full form in a conference proceedings does not have the same
scientific credibility as one published in a peer-reviewed journal
because it has not been through the peer-review process. Even though it
might have been booed out of the hall in the presentation (not saying
this one was) it would still be published in the documented proceedings
of what transpired at the conference.

-- Paul --

The Institute for Neural Cryobiology - http://neurocryo.org
A California charitable corporation funding research to
perfect cryopreservation of central nervous system tissue
for neuroscience research & medical repair of the brain.
Voice-mail: 416-968-6291  Fax: 559-663-5511

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