X-Message-Number: 15538
Date: Fri, 02 Feb 2001 21:39:30 -0500
From: Paul Antonik Wakfer <>
Subject: More comments on old posts - #15451, #15452, #15459, 15461

A few more comments to add to and complete those on posts about
cryopreservation methods made to CryoNet before I resubscribed. 
 
>Message #15451
>From: 
>Date: Thu, 25 Jan 2001 14:24:25 EST
>Subject: more on toxicity etc.
>
>Some misunderstandings seem to persist about toxicity of CPAs and related 
>matters, so I'll try once again to clarify this. If I have made any mistakes, 
>I'll welcome correction, as always.
>
>First, from historical perspective, for many years it was believed that 
>relatively large tissue masses could not be vitrified except with 
>concentrations of CPA so high as to produce unacceptable toxicity. That 
>changed in recent years with the use of CPAs (methoxylated compounds) that 
>were not new in themselves, but were new in the context of new methods of 
>rapid cooling and warming.

Although I am not certain about Mr Ettinger's statement re methoxylated
compounds (I think some of them were entirely new to cryobiology), this
is a good summary of the situation.

>With these, toxicity was reported to be lower than with glycerol

Not merely "reported", but clearly measured by the K/Na ratio (see
below), although not yet published.

>--meaning, I 
>believe, the glycerol concentration that Alcor had for some years been using. 
>That, if memory serves, was reported as 7.5 M, which I believe works out to 
>about 690 grams of glycerol per liter of solution, or roughly 60% by weight 
>or 55% by volume.

There is no need to use memory here, the glycerol concentrations used
were fully documented in suspension reports published in Cryonics by
Mike Darwin before 1992 and later by others (although with far less
documentation). Until the early 90s, the glycerol concentration aimed at
for all Alcor patients was about 4-5 M (related to some very early
cryobiology research). It was raised only during the early 90s on the
basis of some EM examinations of cryopreserved animal brains (if I
recall correctly). It was understood when this increase took place that
toxicity was being increased (and Alcor in their ill-informed enthusiasm
sometimes pushed it far above what was a reasonable goal - to 8.5 M in
one case I think), but this added toxicity was thought (guessed) to be
less difficult to repair by the advanced methods of the future than was
the ice damage which had been seen in the EMs.

The history of this use of glycerol and the reason for a concentration
change was described in a article by Charles Platt "New Brain Study
Shows Reduced Tissue Damage" which first appeared in CryoCare Report #4
July 1995. Earlier studies with 4-5 M glycerol in dogs and cats had
showed much more damage than current studies with dogs reaching a
terminal concentration of 7.4 M with a time course of ramp, from a
starting concentration of 15%, of about 2 hours. This last was the same
protocol being used for humans by BioPreservation at that time.

>A couple of people expressed the opinion that the toxicity must also be much 
>lower than that of the current CI solution, which is 75% glycerol by volume 
>(i.e., 750 ml glycerol added to 250 ml water at room temperature, later 
>refrigerated). However, our one-pass does not result in equilibration. Our 
>measurements indicate a final average concentration in the brain tissue of 
>around 26% by weight, hence obviously a much lower toxicity than the previous 
>Alcor standard.

What Mr Ettinger has consistently given no indication of understanding
is that the toxicity of a perfusate is partially dependent on the
differential concentration between it and the surrounding tissue. That
is why is it necessary to begin perfusion with a lower concentration and
only end with the highest concentration after the tissue concentration
has risen (which is shown by the lowered difference between the input
and effluent concentrations as measured dynamically by a refractometer.
The fact that equilibration (uniform tissue concentration equal to that
of the entering and exiting perfusate) was not reached shows that the
glycerol concentration varied throughout the tissue with an average of
26% (although he has given no indication of how this was measured),
clearly far less than the entering perfusate (at 75%).

>How were measurements of toxicity of glycerol made? I don't know; if someone 
>can direct me to reports on this, good.

EMs of animals cryopreserved with the same protocols as humans were used
as described in CryoCare Report #4 July 1995.

>We do know, from recent reports, that estimates of toxicity of solutions 
>similar to the current Alcor solution (but less concentrated, according to 
>Fred Chamberlain's report in CRYONICS, which would imply that the Alcor 
>solution is more toxic) were made from rat brain slices using the K/Na ratio 
>criterion, which most recently showed 66% of normal function, probably 
>meaning an average over the cells in a sample. 

And these K/Na ratios were far better than with all glycerol
concentrations currently in use in cryonics.
To quote one of the scientists involved:

"What is the significance of CI's electrophysiology experiments?  At
INC,
hippocampal brain slices carefully cryoprotected with glycerol (at a
variety of concentrations), frozen, thawed, unloaded of cryoprotectant
and incubated under physiological conditions showed a K/Na ratio that
ranged between 0% and 5% (+-5%) of control slices. This is a terrible
result, and completely incompatible with successful transplant of a
brain so treated.  If the same researcher (Yuri Pichugin) has obtained
good electrophysiology from brain tissue frozen with glycerol, that is
prima-facie evidence of the unsuitability of electrophysiology alone as
a measure of viability after cryopreservation, just as it was for MTT [a
mitochondrial viability assay].  A viability assay that correlates with
transplantability "trumps" an assay that gives positive results in
tissue that is patently untransplantable."

and:

"Perhaps most importantly, the K/Na ratio test is an accurate predictor
of post-transplant viability of perfused and/or cryopreserved kidneys. 
Organs subjected to protocols that give good K/Na ratios in slice tests
tend to survive transplant.  Organs subjected to protocols that give
poor K/Na ratio tests will not survive transplant."

>I also have the impression-and again will welcome correction if I am 
>wrong--that the K/Na test requires very fresh specimens, virtually no post 
>mortem delay. This means that the relevance to cryonics patients is 
>problematic. In the two Alcor cases that were "vitrified," the delay was 
>reported as more than a full day, and there is small likelihood of any 
>significant number of patients being treated with no delay.

The illogic of this argument against the applicability of the K/Na
ratio as a valid initial method of choosing "best" cryopreservative
solutions has been discussed before and should by now be patently
obvious to all.
 
>This does not mean that I personally think the "viability" criterion is the 
>most important one. My guess is that histology is more important, and 
>neuronal electrical activity the most important of all.

If one is going to rely 100% upon Nanotechnology to repair all damage,
then perhaps histology (ultrastructure), if accurately and properly
assessed, is most important. But if one is aiming toward perfected
suspended animation and/or acceptance by the scientific and medical
community, then "viability" is clearly more important. However, once
vitrification is reached, there is, in fact, no trade-off or contention
between these two criteria, as further study of tissues cryopreserved
with the current 21CM protocols will almost certainly show.

>As previously noted, 
>Pichugin's rabbit brain studies were encouraging in this respect, using 
>glycerol and rewarming the specimens from liquid nitrogen temperature.

Absolutely not! With the protocol used, *both* histology and viability
were extremely poor. I have already stated, as textbooks in medical
physiology clearly explain, that is it quite possible to have good
electrical activity with completely shredded cells.

>Finally, yet again, to my knowledge there have been no reports--anywhere, by 
>anyone, ever--of results evaluated by any criterion after application of the 
>current Alcor procedure to brains, cooling to liquid nitrogen temperature (or 
>even the hoped-for intermediate temperature in the range of - 135 C) and then 
>rewarmed.

As Mr Ettinger well knows and has fully supported, for the benefit of
the dying patient who needs it *now*, we must always use the procedure
which recent evidence has indicated is "best", even though full
verification will only be done later. This has always been the case in
cryonics. It is at the very basis of its ideology. If this was not so,
then we would not be cryopreserving anyone until suspended animation was
fully perfected, just as establishment scientists and cryobiologists
demand. 

>Message #15452
>From: 
>Date: Thu, 25 Jan 2001 14:29:58 EST
>Subject: correction
>
>A small correction: In my earlier post I said we add 750 ml glycerol to 250 
>ml water. I meant 250 ml of washout solution.

Thanks for this important correction. Would you please state precisely
what is the composition of the washout solution.

>Message #15459
>From: 
>Date: Fri, 26 Jan 2001 13:55:54 EST

>Subject: new topic: treating enlarged hearts?Also comments on vitrification 
discussions

[snip]

>p.s., I'm learning a lot via the Ettinger driven vitrification discussions.  
>I'm glad to see a (generally) healthy discourse on the procedures, and I'm 
>especially interested in independent labs verifying the test materials and 
>results.  Even if there have been few standards in the past, or they have 
>varied, would it not be a great idea to have at the next major cryonics 
>gathering a development of protocols/testing defined to enable everyone to 
>measure benchmarks in improvement?  ie, how toxic is some chemical, what 
>concentrations are needed, how successful is the vitrification, how fast do 
>patients need to be cooled, how easy is the application in real world 
>conditions?  And, what are the test procedures a lab uses to verify these 
>results? Also, any particular procedure could be verified for practicality- 
>does it help, does it work, is it likely to make a difference?   It could be 
>called the Ettinger Scale of Cryonic Preservation.  Okay, I'm a bit tongue in 
>cheek, but doesn't it make sense to have a Cryonics Standards Institute, 
>seminar or such meeting?  What is that organization-  The American National 
>Standards Institute?   How do they run or determine procedures?  That might 
>provide a benchmark for a similar effort in the cryonics movement... and, 
>could lead to QUICKER advancements in the application of new technology and 
>development of a true " suspended animation" technique.  Okay, we're up in 
>the blue sky territory now, but its so odd to me that although cryonics is 
>striving towards a goal, that goal is not clearly defined with benchmarks 
>along the way to the goal.

HEYMIKE's suggestions here are so obvious and sane that, that it is
truly an indictment of cryonics and cryonicists that they have not
happened in 30+ years of cryonics history and show no sign of been
capable of happening still. As I recall, some kind of cryonics standards
association or at least umbrella organization was attempted in the 1980s
(by the Chamberlains, I believe) but it did not get very far. In the
1990s, the Prometheus Project was conceived as a non-partisan joint
research effort, and attracted cryonicists from all organizations. But
it remained unsupported by the major cryonics organizations, vigorously
opposed by the largest two, and eventually foundered, although not
entirely since the Institute for Neural Cryobiology has become a kind of
heir to its plans.
Unless the membership *demands* these kind of cooperative goals and
standards, the current leaders of the cryonics organizations are simply
going to carry on with their old competitive, protective approach to
each other.

>Message #15461
>Date: Sat, 27 Jan 2001 08:06:44 -0500
>From: Thomas Donaldson <>
>Subject: answers to 2 questions
>
>   Since I am still officially a member of the Board of INC, Ettinger
>   wanted my opinion on the ionic method to assess viability. The first
>   thing I'll have to say is that although I founded INC, and paid to
>   get it set up as a corporation AND one to which you can donate in
>   the US and pay no US taxes on the income you donate, I'm no longer
>   involved in the discussions.

And we should be eternally grateful for Thomas for setting up INC, going
through all the hassle of gaining charitable status and maintaining it
for almost 5 years until it had a role to play (the original purpose
never came to fruition). Through its Hippocampal Slice Cryopreservation
Project, INC has played a crucial role in the development of
low-toxicity vitrification, and with future support now has every
intention of continuing to make major contributions toward reversible
brain cryopreservation and eventually perfected suspended animation.

>   I would value the ionic method for assessing viability because it
>   does touch on a characteristic of our cell membranes which is 
>   quite necessary for viability.

And its operation requires far more than merely one characteristic of
cell membrane health as I have explained before.

>   However if we can get actual operation
>   of neurons: sending impulses and responding to them, then that's
>   much more valuable and trumps any other method.

Again as I have explained, physiologically this is not so for mere
electrical activity of nerve fibers. Now, coordinated brain waves, that
is another story, but it is not measurable except for whole brains as I
understand it (just as EKG readings would not occur for heart pieces).

-- 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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