X-Message-Number: 2679
Date: 04 Apr 94 16:47:29 EDT
From: Paul Wakfer <>
Subject: SCI.CRYONICS (fwd) Greg Fahy on Cracking
To: CRYONET
-- Message forwarded --
>Date: 03-Apr-94 22:01 PDT
>From: Greg Fahy [72124,1173]
>Reply to: Donaldson & Ettinger Postings
This message is intended for Paul Wakfer
and can be distributed to other concerned parties.
Cracking of Cryopreserved Brains
As I understand it, there seems to be a feeling that cryopreserved brains
do not crack if modern methods of preservation, defined as methods similar
to those in use in Ettinger's laboratory, are employed, and that previous
reports of brain cracking reflect "old" experiments that can be safely
disregarded as being too unrefined to be pertinent. I would like to suggest
an alternative interpretation.
In my experience, cracking can take place under two conditions. First,
if kidneys are treated with relatively low concentrations of cryoprotectant
and cooled to -135 deg. C, they crack quite seriously, whereas vitrified
kidneys do not fracture upon cooling to the same temperature. Second,
vitrified masses do crack upon cooling to liquid nitrogen temperature (minus
196 deg. C) if they are cooled faster than a few tenths of a degree C per
minute.
I do not know the precise conditions of the Michigan experiments, but I
do understand that they involve perfusion with massive concentrations of
glycerol without permitting much time for glycerol distribution and uptake
into the brain. I also understand that brain shrinkage is considered
desirable by the Michigan investigators, and that rapid glycerolization is
done partly for this reason. Based on the above, several possibilities can
be considered.
In the first scenario, Ettinger's brains are vitrifying, but are not
cracking because a) they have shrunken so much that they are no longer in
contact with the skull, or b) they have shrunken so much that their
nominal coefficient of thermal expansion/contraction reflects that of
dry brain matter, i.e., is insufficient to result in cracks, or c) they
have shrunken so much that their total volume is insufficient to summate
thermal stresses to the point where they exceed the material strength of the
brain. The key point here is extreme shrinkage. When I have perfused rabbit
brains under conditions that result in extreme shrinkage (6 M glycerol at
temperatures below about 10 deg. C), the histological distortion caused was
massive, even to the point of altering the staining characteristics (i.e.,
possibly the very chemical make-up) of the brain, even though grossly the
amount of brain shrinkage seen was not all that impressive. In Ettinger's
experiments, the degree of shrinkage should considerably exceed what I
observed, and the question that arises then is: is the shrinkage causing
far more serious injury on a chemical and ultrastructural level than the
fracturing that is being avoided?
The second scenario is that, due to the rapid rate of glycerolization,
there is actually minimal uptake of glycerol into the brain. Under these
conditions, the lack of cracking would relate to the lack of glycerol in
the brain substance. According to Mike Darwin, lamb brains frozen with
no cryoprotectant to liquid nitrogen temperature do not fracture. This
possibility is perhaps even more fearsome than the first, since it
implies essentially "straight freezing" which, according to my observations,
is extremely damaging.
In considering these possibilities, I should point out that, whereas
I found kidneys to crack when perfused with relatively low concentrations
of VS4 (something like 20% w/v -- I'll have to check the exact concen-
tration) and cooled to -135 deg C., I have not found similarly-treated
rabbit brains to crack upon cooling to dry ice temperature; therefore,
it is necessary to say that the cracking properties of brains at
intermediate concentrations have not been well worked out and may be
different from the cracking properties of kidneys, leaving open the
third possibility that Ettinger's brains do contain significant concen-
trations of cryoprotectant but still fail to crack for whatever reason,
though they would not be vitrified.
Regardless of which scenario is correct (including scenarios not
identifiable from the foregoing), the conclusion is the same: until
more details of both the experimental procedures themselves and of the
histological and/or ultrastructural results of such procedures become
available, it is impossible to evaluate the significance of the work. The
work could be highly significant and could indicate a major pathway
forward, or it could be merely a new way of destroying brains. We can
not tell from the information that is available. I would also have to
say the same thing about the Segall experiments showing allegedly good
blood reflow with his proprietary formula vs. poor reflow with some
undefined glycerol protocol. Until histological/ultrastructural docu-
mentation of the results of such experiments, in combination with the
precise details of how the experiment was done, become available, the
results could easily be totally misleading. Just because a dead body
looks a lot like a living body to the naked eye, it does not follow
that the two are in an equivalent state of health. Hence, the naked
eye is not sufficiently discriminating to answer questions of the
kind being asked.
If I understand correctly, Ettinger is planning histological/
ultrastructural experiments in collaboration with Russian scientists
to answer some of these questions. This is exactly what is needed,
and I look forward to the results. In the meantime, the jury is out
on this matter. I only hope that Dr. Segall is following suit.
--- Greg Fahy
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