X-Message-Number: 3940
Date: 01 Mar 95 21:34:10 EST
From: Mike Darwin <>
Subject: CRYONICS Glycerol, Suda and Cracking

Mike Price asks some further questions:

1) Is Suda alive?  I don't  know, especially after the Kobe quake. I do know he
quite elderly and retired some years ago.

2) Why didn't he publish more of his material on brain freezing.  Several
reasons: First, who would publish it?  It was about BRAINS which are not
particularly relevant to the scientific community in terms of cryopreservation.

For a real good laugh, reread the first paragraphs of his NATURE paper as to the
"justification" for his research (i.e., effets of cerebral ischemia!).


Another reason was that much of the work was done for his own entertainment.  It
would not have furthered his career or expanded his CV.  In fact, his career
went along quite nicely.  He spent his later working years in largely
administrative capacities (powerful ones); thus he had little time (let alone
virtually no incentive) to spend the ENORMOUS amount of time required to
prepare, revise, and shepherd through a peer-reviewed manuscript(s) for
publication.  

Freezing brains was of merely academic interest to him by and large; sort of a
hobby; observing that he could do this rather "neat" thing he decided to pursue

it for awhile (this is opinion from me and may not represent how he felt: Thomas
Donaldson and Greg Fahy actually met him and Greg coresponded with him and
talked with (I believe) at some length..

He was incredibly generous with his raw data.  I have a number of slides from
him showing all kinds of neat things which we are only now getting around to
reduplicating in this laboratory.  One lovely slide shows a brain in the skull
which has been coronally sectioned with one hemisphere of normal volume
(unperfused) and the other shrunken following perfusion with glycerol.  He was
also kind enough to provide many slides (photographic 35 mm) of brain histology
after glycerolization, freezing, thawing, storage intervals and so on.  He also

provided hundreds of pages of documenting manuscript and notes; unreadable to me
and most others because it is in Japanese: although I believe Greg Fahy had one
of his Japanese colleagues look over the material for highlights (I could be
wrong here).


One technique I learned from him is the importance of carbon perfusion, which we
are now using routinely here to determine capillary level distribution of CPA
and perfusdion defects related to clotting, embolization, CPA dilution from
extracellular water, and so on.

And yes, it IS incredible work and quite amazing that it worked at all.  If you
like, when I come to England this Spring or Summer, I will bring some of Suda's
slides and also do a presentation on our work with various CPAs, coling/warming
rates and research in general on brain cryopreservation.

Finally, while theory would indicate that more CPA=more protection, this may be
only partly true.  High CPA concentrations are also toxic and cause
ultrastructural changes of their own.  This has been a major headache for us
here: sorting out what is artifact and what is REAL.  We are slowly and
time-consumingly solving the problem by looking at tissue prepared for
examination in many different ways: for instance fixing in the presence of CPA,
fixing after CPA is removed, fixing with CPA after freezing (i.e., matching the
terminal effluent glycerol concentration plus adding fixative), doing freeze
substituition at -90xC, and so on.  None of this work is either easy or cheap.
Currently we are working mostly with dogs because we are exploring the effects
of cryopreservation in the context of current cryonics procedures.  Shortly, we
we will be greatly expanding our small animal work because it is more cost
effective and will be focused on optimum scenarios rather than those involving
some preexisting period of cardiac arrest followed by CPR, TBW and
cryopreservation.

One reason I tend to think slow cooling might be the answer is an observation I

made many years ago with Alcor/TT patients.  That observation was that there was

NO cracking on the backs of the patients, ever.  The backs were better insulated
because they were on stretchers in most cases (= slower cooling rates).

As to your question about the three patients who were autopsied:

1) Only one was perfused with glycerol.  Terminal molarity in the venous
effluent was in the 3M range.  This patient had the worse internal cracking.

2) The other two patients were treated with very low concentrations of DMSO.
Terminal tissue concentrations were 3-8%.  One of the patients had been cooled
to -196, rewarmed to -79xC and cooled to -196xC again, following which he was
thawed (the body).  This patient had extensive external cracking but little
internal cracking.


3) Cooling of these patients was controlled to some degree, but almost certainly
did not use the same profile as CI.  These patients were frozen in the mid to
late 1970's.  Things have chaged since then!  More to the point, their warming
was uncontrolled and rapid with large surface to core differentials.  We tried
to rule out this as a variable with the cats (which were also cracked) done in
the mid-80's by carefully controlling rewarming.  Unfortunately, the cats were
cooled comparatively rapidly from -79xC to -196xC (over a day or two at most)
My guess is that both parameters, cooling rate and rewarming are of
significance.

Hope this answers your questions.  The real answers should come out of the
laboratory both here and elsewhere with further experiments followed up by well
documented reports on the relevant parameters under which they were undertaken.

Mike Darwin

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