From: kqb@cbnewsl.cb.att.com (kevin.q.brown)
Subject: Cryoprotectant Issues (Re: Freezing Damage)
Organization: AT&T Bell Laboratories
Date: Mon, 14 Dec 1992 16:12:01 GMT
X-Crossposted-To: cryonics mailing list

    ------------------------ Forwarded Message ------------------------

> From: Ben Best <ben.best@canrem.com>
> Date: Sun, 13 Dec 1992 19:00:00 -0500
> Message-Subject: cryoprotectant issues


ERRATA: In my previous posting concerning freezing damage I stated that
        DMSO toxicity varies inversely with temperature, when I meant
        that DMSO toxicity varies DIRECTLY with temperature.

    I am delighted that Mike Darwin has chosen to respond to my
"Freezing Damage" posting in such detail. And I am delighted in
anticipation that I can bounce my speculations off of his vast fund of
knowledge and experience. I will therefore address my remarks to Mike.
But because I hope my remarks are of interest to others, I will explain
myself in greater detail than would be necessary if I really were only
addressing Mike.

   Reminder of acronyms:
     CCP -- Cat CryoPreservation study by Darwin, Hixon and Leaf
     DCF -- December 1991 article in CRYONICS magazine by greg Fahy

     I agree that the vision of nanotechnology has lured too many people
into a smug belief that massive autolytic and freezing damage can occur
without destruction of critical structures. But even if Dora Kent's head
had received perfect suspended animation, nanotechnology will be
necessary (although perhaps not even sufficient) to reverse the effects
of Alzheimer's Disease, to undo the effects of aging, and to construct a
new body. If I die, I hope to be very old -- and I will not expect to be
reanimated until the ravages of old age are reparable and reversible.

     I think that the Smith Criterion is more than a "useful first
approximation" to acceptable levels of freezing damage. It was no
accident that Greg Fahy selected 3.72 Molar (27.2%) glycerol
concentration for his DCF rabbit brain experiment. I should think 3.72
Molar should be the theoretical MINIMUM acceptable glycerol
concentration under ideal conditions (and CCP should be evaluated in the
context of its 3 Molar target concentration, knowing this). I agree with
Paul Wakfer that 3-decimal place accuracy is ludicrous under such messy
conditions as perfusion of the brain. Greg's "3.72 Molar" is virtually a
*codeword* for the Smith Criterion. I think 4 Molar would be a suitable
target concentration to fulfill the Smith Criterion under the best
conditions. But applying the Smith Criterion to glycerol implies that
glycerol perfuses perfectly into cell bodies -- while the significant
observed dehydration implies that glycerol is NOT perfusing into cells
well -- and is, in fact, osmotically drawing water out of cells.

   An idea cryoprotectant for the brain will readily perfuse the
blood-brain barrier as well as cell bodies in the grey and white matter.
I think this implies a cryoprotectant that is both hydrophobic
(lipophilic) and hydrophilic (lipophobic) and has a low molecular
weight. This could mean a suspension (in the pharmaceutical sense) like
milk or soapy water. It could also mean molecules having both
hydrophilic and hydrophobic properties, like glycerol and (especially)
DMSO. My advocacy of DMSO is not intended to preclude the use of
glycerol. DMSO [(CH3)2SO] is known to perfuse better than glycerol
[(CH2OH)2CHOH], possibly in part because of its smaller molecular weight
(78 versus 92) and in part because of the lipophilic methyl groups.
Nonetheless, DMSO is a much more reactive molecule. In fact, it is used
as a reagent based on both its ability to transfer oxygen, and its
ability to transfer a methyl group. At lower temperature, however, this
reactivity is greatly lessened and its cryoprotectant properties can be
exploited. For this reason, in freezing 8-celled human embryos, glycerol
is used initially and DMSO is only introduced at low temperature. The
value of DMSO as a cryoprotectant cannot be viewed independently of the
temperature at which it is introduced. Similarly, rewarming tissues
cryoprotected with DMSO for microscopic examination is likely to result
in DMSO chemical activation unless the DMSO is carefully removed.
Nonetheless, Greg Fahy included DMSO in the cocktail with which he
perfused kidneys at above-freezing temperatures (glycerol excluded),
with 100% viability. How can this be accounted-for in light of the other
evidence?

   If DMSO causes interstitial edema in ischemically injured patients,
then it shouldn't be used for such patients. But why deprive others of
possible benefit? Shouldn't suspension protocol be tailored to the needs
of the patient?

     Suda's reference in BRAIN RESEARCH 70 (1974), p527-531 is to a
paper "In preparation" which (as far as I can tell by searching the
CITATION INDEX) was never published. (I think Suda was at the end of
his career -- maybe he even died before publication.) We know nothing
of his methods in comparing DMSO with glycerol, and we know lots about
his shortcomings in the experiments he did publish (relatively low (15%)
glycerol concentrations, inadequate washing of glycerol on re-warming,
failure to include nutrient in the perfusion fluid, etc.). Even if it
is true that glycerol is superior to DMSO under all conditions, it might
also be true that both glycerol and DMSO in combination is superior to
either one alone.

     I don't mean to sound like I am stidently advocating the use of
DMSO. My objective is clarification, more than advocacy.

     Please explain what you mean when you say that the blood-brain
barrier (BBB) can be "osmotically opened". I noticed that CRYONICS
magazine reported a 6.02 Molar glycerol concentration in the Glennie
suspension. What was the glycerol concentration on the cerebral side of
the BBB? If glycerol is not perfusing cells, isn't there a danger that
such high glycerol concentrations are simply causing more dehydration
damage?
         Greg's biochemical content was on DCF page 14, upper left
corner.
                -- Ben Best (ben.best%canrem@uunet.ca)
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