X-Message-Number: 4750
Date: 09 Aug 95 15:58:25 EDT
From: Mike Darwin <>
Subject: CRYONICS Fixation & Rewarming

Joe Strout posted some suggestions and ideas about fixation and subsequent
cryopreservation.  There should *definitely* be a part in the FAQ which deals
with this issue because it comes up regularly and is tedious to answer over and
over again.

First, this is not  a new idea.  It has been around for at least 15 years and
was the preferred scenario in Drexler's ENGINES OF CREATIONS.

Second,  Joe is quite right about fixation making subsequent cryoprotection
problematic.  In fact, I'll bet Joe might actually have fixed a few brains or
animals by now.  Aside from protocols based on fixative volumes needed vs.
specimin size, a well known way to tell when its time to stop perfusing with
fixative is when the vascular resistance begins to climb precipitously.
Fixation alters capillary structure and function to essentially bring flow to a
halt.

Third, any neurophysiologist or electron microscopist worth his/her salt will
look at you in horror if you propose fixing an animal via perfusion after say,

even 20-30 minutes of normothermic ischemia.  There are two reasone for this: a)
Many artifacts will appear (such as injured mitochondria and clumped chromatin)
which  could cloud the issue of where the injury came from (i.e.,the
experimental procedure being evaluated, or from ischemia).  This is not the
major concern for cryonicists.  b) Relatively modest period of ischemia result
in very poor distribution of the fixative.  Red cells become rigid when ATP
depleted and stick in capillaries, I-CAM is activated causing leukocyte and RBC
plugging, and regional cerebral blood flows go to hell through loss of normal
vasomotion.  Prolonged, low pressure cryoprotective perfusion seems to open up
many capillary beds which would remain closed during fixative perfusion.

Of course, it could always be argued that fixation could FOLLOW
cryopreservation.
We have found this problematic in that the very high viscosity of the perfusate
inhibits fixation, as well as probably cryoprotectant induced alteration of
proteins in ways that make them less likely to go through the condensation
reaction wherein the hydroxyl and amine groups on proteins react (by
crosslinking and producing water as a byproduct) and become "fixed."


Another point of great importance needs to be made here as well, and Joe alludes
to it.  Aldehyde fixatives fix proteins.  They do not fix carbohydrates or
lipids.  While carbohydrates may not be very important to memory, identity, and
so on from an information-theoretic standpoint, it would be hard to argue that
lipids are equally inconsequential since they comprise the major load of the
membrane structure which is both the identifying border of cells and the locus

for many, many critical biochemical reactions which undoubtedly are important to
identity.

In order to fix lipids you must generally use a metal like osmium in a reactive
form (such as the tetraoxide).  Osmium is not, I repeat not,  nice stuff.  You
might be able to substitute mercury compounds or even arsenicals, but the
resulting mass of tissue would be not only mildly poisonous, as is the case
after aldehyde fixation, but highly poisonous and have a top-drawer haz mat
classification.  In fact, the use of heavy metals for embalming corpses is
specfically forbidden by law in many states (maybe all?) in the US for two
reasons: danger to the ground water and environment and, more appropriately,

because salts of  metals like arsenic and mercury make such good poisons for the
purpose of rendering otherwise healthy (or at least not immediately terminal)
people dead.  This is called homicide.

A major reason for the deterioration Joe spoke of in fixed materials is that
MOST of the specimin is not fixed.  Anyone who has stored a human or animal
brain in a jar of aldehyde fixative will observe the progressive leaching of

compounds into the bathing media and the progressive deterioration of the tissue
macroscopically over a period of several years.  Indeed, in specimins where the

fixative level was allowed to drop exposing the specimin in a closed jar, I have
observed a white, fuzzy mold growing quite nicely on such tissues -- presumably
in a formaldehyde saturated atmosphere!


Finally, what no one in this discussion has considered is the effect of fixation
on cryoinjury.  My expectation was that it would reduce it since everything
should be turned into a rubbery polymer.  Certainly cells in culture which are
fixed and repeatedly frozen don't dissolve away like unfixed freeze-cycled
controls.

However, one nasty consequence of fixation is that it reorganizes membrane
structure and opens large pores in the plasmalemma.  These pores allow free
movement of water, cryoprotectants, salts, and even fairly large molecules like
mannitol (MW ca. 190).  As a consequence, cells do not dehydrate when freezing
occurs and ice propagates right across the cell membrane resulting in
intracellular freezing.  EM examination of such tissue is a dismal business.
The tissue looks WORSE than if it had been straight-frozen without any
cryoprotectant or fixative at all.

Finally, as alluded to earlier, fixation requires the *diffusion* of chemicals
and for diffusion to be effective and rapid enough to inhibit autolysis the
maximum distance from fixative to tissue in need of fixation can be no greater
than 1 mm and preferably no more than 0.5 mm.  In many, maybe even most of
today's patients, you are not going to get good distribution of fixative,
period.  Cooling, on the other hand, while slow in large masses, still will
eventually reach ALL of the tissue and in a far more timely manner than
fixation.  Its effects are thus independent of the condition of the circulatory
system or distances between the surface and core of an un- or underperfused
area.

As to heating, I see no need for little pellets full of nasty, highly reactive
compounds. Existing RF technology can rapidly and uniformly rewarm masses the
size of human kidneys in excess of 300 to 400xC per minute.  Theoretical
consideration and some clever design work have demonstrated that the limits of

RF heating are not particularly mass dependent -- at least not as far as a human
body is concerned.  Whole humans can, in theory at least, be rewarmed at
comparable rates even with the very limited consideration given to this problem
so far.  Adequate cryoprotection, present either before or after repair would
make this problem much easier by eliminating the presence of ice.  Such
cryoprotectants would also serve to greatly relax the requirement for very high
rates of rewarming to avoid ice formation after repair, from thousands of
degreees C per minute to hundres of degrees -- or even lower.

I find I must agree with Charles; some of these scenarios, given the
technological capabilites they posit, certainly are Rube Goldberg ways of
achieving what might simply be done by other means such as using the same

end-stage nanotechnology in combination with simple, macro approaches such as RF
rewarming.

Ralph's scenario and the background which produced it  remind me of the old
adage: When all you have is a hammer, every problem looks like a nail.

Mike Darwin


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