X-Message-Number: 1410
Date: 09 Dec 92 02:18:22 EST
From: Mike Darwin <>
Subject: CRYONICS: Reply to Ben Best
From: Mike Darwin
Re: Ben Best's remarks on cryoinjury
Date: December 8, 1992
Ben Best's response to my posting regarding ultrastructural
damage was a joy to read. Ben has asked many good questions. Some
there are no answers for (that I know of) at this time. However, some
of the issues he raises have been the subject of investigation and I
would like to respond.
1) The Smith Criterion was never meant to be anything but a
useful first approximation to guide us in determining how much ice
formation to "accept." In particular, it does not speak to the unique
situation posed by the presence of cryoprotective agents and cooling
to deep subzero temperatures. Smith's hamsters were cooled to a core
brain temperature of -0.5 degrees C with *no cryoprotection*. This is
a far cry from -79 degrees C and multimolar glycerol. I have the same
reservations about flatworm and salamander experiments designed to
"prove" memory conservation after freezing. These ARE useful
experiments, but our optimism about them should be guarded at best.
2) Why did we choose 3M glycerol for this study? Well, because
that was what we were using in humans at the time. When this study
was begun (nearly a decade ago) there was relatively little experience
with closed circuit perfusion and high terminal glycerol
concentrations in human patients. Indeed, it wasn't until Terri
Cannon was perfused in 1987 that the "current" system of closed-loop,
dual reservoir perfusion was used. I might also add that until that
time, most patients rolled in the door many hours after cardiac
arrest: clotted, in full rigor and barely perfusable. We felt elated
to get 1M CPA in! Ben, you have no idea how much things have changed
in the past decade. Also, keep in mind that this was nearly a decade
ago, and most organ cryopreservationsts who were using glycerol (or
DMSO for that matter) were using concentrations in the range of 3M.
Toxicity was a major concern (more on that later).
3) Why not use DMSO. Plenty of reasons. First of all, we have
repeatedly compared light micrographs of brains treated with DMSO vs.
glycerol in the same concentrations. The glycerolized brains look
better -- by a lot. Second, both Greg Fahy and I have some experience
with high concentrations of DMSO (he with kidneys and brain slices, I
with brain slices). DMSO resulted in poorer structural preservation
and foaming of the carrier solution (indicating elution of proteins)
during its removal.
But more to the point, DMSO is simply unperfusable in
ischemically injured patients. They very rapidly develop massive
interstitial edema and this brings perfusion to an early halt. It's
too bad Jerry Leaf isn't around because he could add some first-hand
horror stories on this account since his first few patients were
perfused with DMSO.
4) Secondly, there is the issue of DMSO's penetrability. While
the dehydration observed with DMSO isn't as severe, it is still
serious (skeletal muscle, tongues turn to leather, etc.).
5) Greg has perfused dog heads with his vitrification solution.
The results in terms of brain ultrastructure were disappointing. Once
again, glycerol perfused brains appeared better preserved. The above
notwithstanding, I feel that studies perfusing VS4 in brains should be
repeated as soon as possible. The early studies were conducted with
dog heads shipped hypothermic from California -- far from ideal!
6) You have to rewarm if you want to see any ultrastructure. You
really need to take a firsthand look at EMs after freeze substitution:
all the structure is *dehydrated*. Trying to evaluate it in the
dehydrated state is like trying to read a book CLOSED. There is no
detail visible. Period. In fact, one of the major problems with CCP
is that we can't be sure what structural changes were a result of
glycerol dehydration and which are the results of cryopreservation. I
will be starting work to answer this question after the holidays.
Specifically, I will be perfusing brains with glycerol and *then*
fixing them (with glycerol still present) in the absence of freezing.
I will also be deglycing brains and fixing them. It should be
interesting to see if this is possible without developing massive
cerebral edema.
7) In Greg's freeze-substitution studies osmium fixation was
carried out at -79 or lower. Rewarming was AFTER fixation and was
carried out to allow Epon embedding and and sectioning.
I know of no studies done by Greg which evaluated histochemistry.
I would also point out that Greg repeated some of our work with
rabbit brains perfused to 4M glycerol and got similarly bad results
using a different animal model and a different EM technician.
8) Regarding DNA being a storehouse for memory. I don't buy
this. This doesn't mean it isn't so, but it is not widely accepted
and I have heard some arguments that (at the time) seemed convincing
in opposition to this idea. In fact I heard them from Greg Fahy.
The point is, *we don't know memory is stored*. Therefore we
want to try for as great a degree of preservation as as possible.
Chopping our brains to ribbons with ice is NOT, I think we can all
agree, the best way to achieve this.
Finally, I would like to say that I am sick to death of all this
hand waving about possibilities for repair. It is a fool who spends
all his time trying to repair the results of an accident that could
have been prevented in the first place. In my darker moments I almost
wish that cryonics had never been thought of and that all the time and
money that has been spent on it would have spent on solving this
problem. The problem of injury-free or modest injury cryopreservation
is NOT, I repeat NOT the most difficult problem in the world to solve.
As Ben points out, what we need are some fairly basic studies to
determine which cryoprotective agents are the best for brains, and
then we need to do brain perfusions with multimolar concentrations of
the agent(s) and greatly reduce or altogether eliminate ice formation.
This is work that could have been done 20 years ago! Indeed, it was
work we started to do over a decade ago. CCP was a survey project
designed to tell us what we needed to do. It succeeded in this
respect: we failed in following through.
Cryonics is full of sterile arguments about how many angels can
dance on the head of a pin. In the long run a lot less effort and
anxiety will be expended if we just solve the goddamn problem of good
preservation in the first place.
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