X-Message-Number: 1404
From: Ben Best <>
Date: Sun, 6 Dec 1992 19:00:00 -0500
Subject: freezing damage


                 FREEZING DAMAGE: TRAGEDY AND HOPE

    My reaction to Dr. Merkle's article on the technical feasibility
of cryonics is very similar to that of Hal Finney: What good is
nanotechnological repair capabilities if the structural basis of
memory and identity is destroyed by cryonic suspension procedures? I
wrote Dr. Merkle a letter to this effect, but now that Mike Darwin
has posted his cat cryopreservation studies to the net, I want to
let loose with some comments, some of which have been brewing for
over a year. I shall designate my primary references by three TLAs
(Three Letter Acronyms): CCP, the Cat CryoPreservation study by Leaf,
Darwin and Hixon; DCF, the December 1991 article in Cryonics magazine
by Greg Fahy; and FCR, the Funding Cryonics Research comments in
CRYONET messages 484, 485, 486, 490 and 498.

   As background, people should understand that the "Smith Criterion"
has been central to cryonic procedures for many years. In the 1950s,
Audrey Smith froze hamsters at temperatures not far from 0*C and
achieved complete recovery of hamsters whose brains had been 60% (but
not more) ice. The explanation for this is that with slow freezing, ice
initially forms in the extracellular spaces and initially causes no
damage. A 68% concentration of glycerol in water will vitrify (harden
like glass) rather than form ice crystals at all temperatures down to
liquid nitrogen (and below). But this solution is far too viscous to
be used for cryonic suspension purposes. But based on the "Smith
Criterion" that 60% ice formation in the brain will do no neurological
damage, a 27.2% (3.72 Molar) glycerol concentration is deemed adequate
to keep brain ice below 60% right down to liquid nitrogen temperature.
In theory, ice crystals would form extracellularly while cell bodies and
synapses were vitrified.

      Given this concern with the "Smith Criterion", why did the CCP
study only attempt to achieve a 3 Molar target glycerol concentration?
In any case, the evidence seems clear that perfusion fell below even
this level -- massive dehydration was observed. It has been known for
years, as the CCP paper acknowledges, that glycerol does NOT perfuse
cells well and causes fatal dehydration of the brain at room
temperature. WHY, then, has glycerol been used as a cryoprotectant
in cryonics rather than DMSO, which has better perfusion properties? I
have asked this question repeatedly and the only answers I have heard
is that DMSO is more expensive and more toxic. Yet DMSO could be
introduced at lower temperatures, since its toxicity varies inversely
with temperature (and this is the procedure used in freezing human
8-celled embryos ot liquid nitrogen temperature). Moreover, the
mechanisms of cryoprotectant toxicity are unknown and may involve
nothing more than enzyme denaturation. If DMSO produces no STUCTURAL
damage, the effects of toxicity may be more easily dealt with by
reanimation technology than the damage to structure seen with glycerol.
But in FCR, Greg Fahy reports 100% recovery of kidneys loaded and
unloaded with a DMSO, formamide and propylene glycol cocktail at above-
freezing temperatures.. Greg also says that he could customize a
cocktail for cryonics suspensions to "reduce ice growth enough to
preclude structural damage". Can there be any higher priority to
cryonics research than to find a suitable replacement for glycerol? But
why isn't DMSO being used NOW, given present knowledge?

    Greg Fahy used 3.72 Molar glycerol in his DCF electron microscope
study of rabbit brains. He refers to remarkable tissue re-organization
and complete recovery of biochemical function upon thawing, which seems
to contradict the evident ultrastructure damage. CCP acknowledges that
thawing damage, sectioning damage and fixation damage may have
contributed to the ultrastructure damage observed. (And we can add
dehydration damage and damage due to an inadequate target
concentration.) FCR makes reference to Greg Fahy also rewarming and
fixing with osmium tetroxide. Which raises the question, how much of
this observed damage is really FREEZING damage? And why should
rewarming be necessary when the method of choice for neurohistological
ultrastructure studies is frozen microtome slices rather than chemical
fixation?

    Given the superior ultrastructure preservation observed for the
kidney and heart in CCP, have studies been made or how well
cryoprotectants cross the blood brain barrier? Sodium lauryl sulfate
is used to improve formaldehyde perfusion, maybe it could assist
cryoprotectants as well.

    The final question I want to address is: What is the structural
basis of memory and identity in the brain? As Thomas Donaldson has
repeatedly alluded-to, long-term memory seems to be similar to
embryological differentiation. Long-term memory seems to be associated
with modification of synaptic connections and (perhaps more importantly
for cryonicists) with modification of the neuronal DNA that programs the
synaptic connections. The implication of this "redundancy" is that
damage to synapses might not matter if the information encoding those
synaptic connections is preserved in neuronal DNA. Future nanotechnology
could reconstruct a brain from neuronal DNA alone, if this is true.
Neuronal DNA appears to be very robust -- many tissues (including
neurons from aborted human foetus' used for Parkinson's patients) have
been frozen in liquid nitrogen temperature without losing tissue-type
differentiation. My understanding is that the damage Greg Fahy observed
was EXTRACELLULAR damage, meaning that neuronal DNA may have been
mostly preserved.

   Damage does not mean destruction. Even current cryonic procedures may
not be destructive. And titanic efforts may not be necessary for
significant improvement.
                         -- Ben Best (ben.best%)
--
Canada Remote Systems  - Toronto, Ontario
World's Largest PCBOARD System - 416-629-7000/629-7044

Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=1404