X-Message-Number: 2676
Date: 03 Apr 94 23:58:48 EDT
From: Mike Darwin <>
Subject: SCI.CRYONICS fracturing

At the risk of sounding like a broken record I wish to 
second Thomas Donaldson's observations on the need for more 
research.  As regular readers of Cryonet and Sci.Cryonics 
will probably have noticed, Thomas and I seem to have a 
regular mutual admiration society going on this point!

I also wish to respond to remarks made by Bob Ettinger 
regarding both cracking and Cryonics Institute (CI) and 
Immortalist Society (IS) Research.  Bob remarks that CI does 
not observe cracking and that the comments about the 
existence of cracking are "probably as a result of old Alcor 
research."  In this he is partly right; the work done by 
Darwin, Leaf and Hixon some years ago at Alcor was the first 
to point out the existence of this phenomenon to the 
cryonics community and the first to document it in 
cryopreserved humans.  However, the fact that it is "old" 
research should have little to do with whether or not it is 
"good" research or "valid" research.  Nor do Bob's 
statements about CI's results with sheep heads lay the 
cracking issue to rest.

But first a little background.  We were not the first to 
observe cracks either in bulk solutions or in organs loaded 
with multimolar concentrations of colligative 
cryoprotectant(s).  This phenomenon is reasonably well 
documented and was first observed in bulk solutions by 
Kroener and Luyet in 1966 (Formation of cracks during the 
vitrification of glycerol solutions and disappearance of the 
cracks during rewarming. Biodynamica 10:47, 1966).  While I 
do not have the reference handy Rapatz reported fracturing 
in frog hearts cooled to -196C during *rewarming* after they 
had been loaded with 13M ethylene glycol.  Similarly, Fahy 
and Sauer et al., have reported extensively on fracture 
formation in concentrated cryoprotectant solutions cooled to 
below their glass transition points (Tg) (Cryobiology 
27:492, 1990).  This problem also occurs in heart valves 
cooled rapidly from -150C to -196C by immersion in LN2 
(Adam, et al. Cryobiology 27:605, 1990).

Work I conducted using cats with Leaf and Hixon at Alcor and 
work I did using rabbits for Soma, Inc. in Indianapolis, In. 
consistently demonstrated fracturing in many organ systems 
including the brain and spinal cord.  Similarly, examination 
of humans perfused to a tissue concentration of 1M DMSO and 
3M glycerol demonstrated a similar pattern of fracturing 
(although the brain was not examined in these patients) 
(Federowicz, et al., Cryonics 5:16,1984).

Recent work by Fahy wherein rabbit kidneys were perfused 
with 15% VS4 and slowly cooled to and rewarmed from about 
10C below Tg (to -135C) disclosed the presence of large 
fractures as assessed by reperfusion with plastic and 
corrosive dissolution of the organ to reveal a plastic 
vascular cast (Greg Fahy, personal communication).

So how do we reconcile these results with Ettinger's 
seemingly contradictory results?

Part of the problem is that Bob has never published any kind 
of detailed report on his work.  In order to evaluate the CI 
results we need to know under what conditions the 
experiments were conducted.  What was the approximate 
concentration of glycerol in the tissue as determined by 
measuring the glycerol concentration in the venous effluent 
(this is simply done with a hand-held refractometer)?  What 
concentrations of agent were perfused in and what was exact 
composition of the vehicle solution?  And very importantly, 
what were the cooling rates and warming rates?

The CI model as I understand it from the incomplete reports 
Bob has written for the IMMORTALIST differs in some 
important ways from work by done Alcor and others -- ways 
which might explain why CI did not observe cracking. 

One important difference was the immediate perfusion of very 
high concentrations of glycerol (50% to 70%).  This would 
result in severe cerebral dehydration pulling the brain away 
from the skull.  Another potentially critical difference is 
the opening of a large window in the skull to facilitate 
observation of the brain during perfusion.  Both Greg Fahy 
and Biopreservation have demonstrated that it is possible to 
cool brains to LN2 temperature (after vitrification) without 
cracking providing they are freed from any point of contact 
with the container they are in and that they are cooled 
slowly.  However, this has little relevance to the model of 
human beings being cooled to below Tg with brains still in 
their heads and still connected to their spinal cords.

Not knowing the terminal glycerol concentration in the CI 
brains is another serious problem in evaluating the 
research.  Both work conducted here and at Alcor has 
demonstrated that animals NOT perfused with cryoprotectant 
develop very few fractures even after cooling to LN2 
temperature.  In fact, fracturing appears to get worse as 
the concentration of glass forming cryoprotectant increases.

The conditions under which Alcor patients and the Alcor 
animal research were carried out have been exhaustively 
documented in both published case histories, autopsy reports 
and in animal research.  In fact the text of the cat 
research paper was posted here (I believe nearly two years 
ago) in messages 1389 through 1392.  Until we see a 
similar level of documentation from CI it is impossible to 
know what to make of their results.

I would urge Bob to carefully document what was done.  The 
fact that CI did NOT observe fracturing is potentially very 
important.  But before we can know that, it will be 
necessary to reduplicate the work.  And before we can do 
THAT we must know the conditions under which it was done.

Currently we (Biopreservation) have two dogs at -90C which 
were perfused under conditions virtually identical to those 
employed by us (and Alcor) on human cryopreservation 
patients.  These dogs were perfused to 6.5M glycerol under 
tightly controlled conditions (using a gradual addition 
system for introducing the glycerol).  They were then cooled 
at a rate of about 4C/hr to -90C.  We hope to take delivery 
on our whole body aluminum pods in the next few days and 
then begin cooling these dogs to -135C.  After this we plan 
to rewarm the animals and reperfuse them initially with 
substrate bearing perfusate and finally with fixative so 
that we can do ultrastructural and histological evaluations.  
In other words, we plan to do exactly the kind of work Bon 
has commissioned in Ukraine.

I applaud CI's decision to commission work in Ukraine and to 
attempt outside verification of their results.  I would urge 
them to publish complete documentation once this work is 

While in no way trying to discourage the work CI is doing 
with Ukrainian scientists (I think such work is worthy and 
deserves support) I would point out that work can be done 
here quite cheaply too.  I can get electron microscopy and 
histology done on an animal for $200.  The advantage here is 
that I am working with a top flight technician whose 
shoulder I can look over as the results come in.  I would 
also point out that work done at Creative and BPI is done 
for ZERO labor costs and done in an environment using state- 
of-the-art equipment and more to the point the exact 
techniques and personnel used to carry out human 
cryopreservations.  Right now this work is not being 
supported at all by the cryonics community (unless you call 
me, Paul Wakfer, Saul Kent, Steve Harris and a few others 
*the cryonics community*!).  

I guess what I'm saying here is: by all means send CI money 
to support starving Ukrainian cryobiologists and get 
important cryonics research done.  But by all means send us 
starving American cryobiologists some money too!

Hopefully the next few BPI Tech Briefs will report on blood 
gas and cardiac output data obtained from the three dogs we 
have "transported" using Thumper support and then perfused 
with cryoprotectant and frozen.  Once we have gross, 
histological, and ultrastructural data on these animals we 
will report it here as well.

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