X-Message-Number: 2923
Date: 19 Jul 94 22:06:09 EDT
From: Mike Darwin <>
Subject: SCI.CRYONICS Re: Discussion

Bob Writes:

<<There are several reasons why in years past I often didn't pay close
attention to the various reports on suspension procedures.

First, my impression was that the activity was often frenetic and impulsive,
with not infrequent reversals of field. (e.g. glycerol to DMSO and back to
glycerol.) This is not to denigrate the heroic efforts of Jerry, Mike, and
others, but we have to strike a balance.>>

Bob has made this assertion before and I don't understand it.  As far as I know
the only switch from glycerol to DMSO occurred nearly 20 years ago and calling
it a switch back and forth  is hard for me to understand since *both* were in
use prior to that time.  CSNY used glycerol for all its cases that I know of,
and Bob Nelson used (mostly) DMSO.  The Chamberlains used DMSO on their first
case with Alcor, and DMSO was used for one or two subsequent cases by Jerry
Leaf.  I don't recall what the reasons for DMSO being used at that time were,
but I believe because it was more penatrative.  I know that TT used DMSO on a

couple of its early patients, I used it on on one I did (RM) and I believe Jerry
Leaf used it on two patients he did: KVM, and Sam Berkowitz.  The one universal
finding in these cases was that  most of the patients (who had also had long
ischemic periods) experienced tremendous visible edema (swelling). Jerry
abandoned it for this reason, as did I.  Since that time virtually *all* Alcor
and most early TT patients (i.e., those done by Jerry Leaf after KVM and SB)

were treated with glycerol.  Thus, as far as I can tell, DMSO was in widespread,
indeed uniform use on the West Coast until a switch was made by Jerry Leaf (and
me) in the late 1970's.  No Alcor patient that I know of has been done using
DMSO since.  I do not know what Segall and Trans Time use, but to my knowledge
(hearsay)  it is not DMSO or DMSO-based. Maybe Art Quaife can provide some

comments.  In any event, there have been very swings in protocol and I would say
that any examination of the case histories published will show that the basic

protocol has been quite stable for nearly 15 years with the following two trends
occurring (and not being reversed):

1) Consistently high concentrations of agent being used.

2) Longer cooling times from -79C to -196C being used.


It was not my intention to be rude (i.e., not exercise ettiquette) in my posting
to Bob.  These are the kinds of questions and responses which are common to the

net, and I generally believe that comments should be made publically in response
to public postings.  I have repeatedly raised these issues before, although
perhaps less bluntly (i.e., more tactfully).  In trying to be tactful in other
areas of my post I may have tarred Bob with accusations which he was not
properly deserving of.  When I spoke of unsubstatiated claims I was speaking
more to material circulated Sternberg and Waitz -- and I should have said so,
especially since most of the post concerned responding to Bob's post.  Bob has
usually qualified his posts about claims in ways which are appropriate.  What I

was unhappy about what was statements which simply didn't reflect the facts such
as the slow cooling rates generally used by Alcor, the previously disclosed
absence of major marco fractures in unglycerolized frozen-thawed animals, and
so on.

Nevertheless, I find it surprizing that Bob would take a position such as is
indicated by his quote: " There are several reasons why in years past I often
didn't pay close attention to the various reports on suspension procedures."  
I pay close attention to *everything* I can related to cryopreservation
procedures.  Being uncertain about things makes such scrutiny all the more
necessary.  I will be tickled pink if it turns out that the cracking problem is

soluable by simply cooling very slowly!  I have to confess that I *like* storage
in liquid nitrogen.  It is neat.  It is very safe.  It is very aesthetic to see

people submerged in this wonderful liquid. Above all it is *easy*.  I would like
nothing better than to find a way to continue to use it.

As Bob goes on to note:

<<Over all, it was just the usual cost/benefit estimate. No procedure was even
claimed to produce assured significant benefits at the bottom line, so why
divert much badly needed resources of time and effort?>>


Well, I guess that depends upon whether or not you think these things can make a
difference. I don't think you get to the point where you are making a big

difference until you start out in the land of the marginal.  For instance, it is

my understanding that Bob picked his very slow cooling rates on the basis of the
early results showing cracking.  Those results were discovered by dint of a

strong desire on my part (and the part of others such as Jerry Leaf) to find out
what the bottom line was.

Bob states: 

<<As to Dr. Harris' comments on tradeoffs and cooling rates, we are glad to
have this input. The answer to his question is that we do NOT take a long
time at the freezing plateau, even though we take a full week to cool to dry
ice temperature (7 days with human patients--5 days for sheep heads). To get
the patient well into the sub-zero Centigrade range takes only about 8 hours.>>

While Steve certainly made the comments about the tradeoffs, it was I who asked

the question abourt how long its takes to go below freezing for CI patients.  It
would help if you could be more specific here principally:

1) What is the likely freezing point of a CI patient after perfusion (in other
words what is the terminal venous glycerol concentration)?

2) When you say: "To get the patient well into the sub-zero Centigrade range
takes only about 8 hours" what exactly do you mean by this, are you speaking of
rectal or esophageal temperatures or are you speaking of air temperature around
the patient?  And at what rate does the patient pass trough the freezing point
-- in other words how long does the patient remain at his/her freezing point.
Again, specific data would be very helpful here.  The shape of the cooling
curves is likely to be important.

Going on to discuss autolysis Bob says:

<<I also note that Mike Darwin's pessimism about autolysis does not seem to be
shared by Ralph Merkle. Further, Greg Fahy's affidavit (1988) contains
references to considerable work suggesting that brains do NOT deteriorate
under warm ischemia as rapidly or badly as Mike seems to think. With both
humans and other mammals, even after several hours of warm ischemia, followed
by freezing and thawing, many indices of physiological activity are rather
good, and often histology also.>>  

I am certainly less sanguine than Bob appears to be about ischemia.  But I was

not referring to warm ischemia in my post, but rather to exposure of patients to

long period of cold ischemia  (neart 0C) in the presence of  high concentrations

of cryoprotectant after the autolytic cascade is activated in warm ischemia.  It
may well be that the cryoprotectant confers protection in such a situation.  I

don't know, and I would prefer not to find out the hard way.  It is important to

point out that we are not talking about "flash" freezing here in any event.  The
relatively "rapid" rate at which we cool our patients is still only 4C per hour
which is incredibly slow by the normal cryobiological standards for cell

suspensions of 1C per minute.  I have tried slower rates (2C per hour) but don't

see any material difference in ice damage -- I haven't looked for differences in
ultrastructural injury).  I have sought to minimize long exposures at high
temperatures to multimolar concentrations of glycerol because of toxcicity and
autolysis.

I am well aware of the literature as it relates to ultrastructural stability

versus ischemic time.  Indeed, a fair number of the papers Ralph (and Greg) cite
were provided by me!  Part of the problem with these studies is that they may
very well be like aerial photos of a city treated with nerve gas.  Everything
looks great -- except for a few cars which have run into bridge abbuttments or
off the road,  everything looks structurally intact.  What the pictures can't
see is  all the dead people.  Certainly synapses and membrane structure look

reasonably good after an hour or so of normothermic ischemia.  And this *should*
give us reason for real hope.  And depending upon what we are (i.e., what our

identity is) it may be all that is needed.  But I suspect that memory is encoded
a layer or two deeper in structures like receptors or ion channels which are
invisible to to EM.  And it is not at all clear that those things are being
conserved.  In fact, there are some human brain injuries which result
(apparently) in permanent loss of declarative memories.  These injuries, quite
disturbingly in my opinion, are all of a kind which result in prolonged whole

brain inflammation and activation of phospholipases and other membrane degrading
enzymes.

The situation with children who experience cold water drowing is not quite as
cut and dried as it first might seem.  Such children chill rapidly because they

have more favorable surface to volume ratios, thinner skulls, and vessels closer
to the surface of the body.  Young animals also have greater intrinisic
resistance to ischemia (probably for metabolic reasons) and respond with the
mammalian diving reflex (a metabolic cost cutter) more frequently and more
profoundly than do adults.  Yet another often overlooked factor is that we do
not really know the arrest time in these kids.  In the dog lab we have all
watched with amazement as the occassional animal keeps a heartbeat down to 11C!
Further, I've turned off the ventilator and had an *occassional* dog keep up a
perfusing ryrthm (i.e., generate an arterial pressure wave) for 15 minutes at

normothermia!  Some of the kids may be profoundly bradycardic during the initial
30-40 minutes of immersion -- during which time they are still being perfused
*and they are also cooling* -- further reducingtheir metabolic demand.

Cryonics patients aren't usually in any way in a comparable situation.  Take
Jerry White as an example (here I really wish I had the ability to show his
graphed data).    He was in deep shock with poorly responsive pupils for more
than 24-hours before he arrested.  Seventeen hours before he arrested his LDH
was 457 U/L (normal range is 0-250 U/L).  By 80 minutes after his arrest (on
Thumper support) his LDH was 3267 U/L.   Based on data from Fried and other

patients I can tell you that probably at least half of the rise from 457 to 3267
happend *before* cardiac arrest occurred.  And if you correct for hemodilution

the LDH continued to "rise" (show evidence of ongoing release) every step of the
way throughout initial washout and right up until the end of cryoprotective
perfusion.  This is indicative of cell lysis or profound alterations in some or
all cell membrane permeability.  This is not the kind of thing a healthy child
submerged in ice cold water is subjected to.  My point here is that the cascade

of ischemic injury is already well underway in the typical slowly dying cryonics
patient.  This is not the case in laboratory studies of ischemia which are

usually focused on sudden-death models and clear start points and do not include
long periods of hypoperfusion and activation of the immune/inflammatory cascade
hours before circulation ceases.

These are very different experiments.

The point here is that we may not even see ischemic injury in this select
population of statistical outlyers (children drowned in cold water)  such as we
would see in an adult subjected to an hour of normothermic arrest, let alone a
an adult subjected to hours of shock and hypoperfusion before his/her hour of
cardiac arrest.

And again, I'm not saying that such situations are hopeless, but rather that  I
prefer to avoid them and that I prefer to minimize injury every step of the way
where it is reasonable to do so. As to what constitutes *reasonable*?  Well,
here men of good will and of good reason may differ.


Finally Bob says:

<< I suggest that "Smith's Criterion" is a very weak reed on which to
lean. For one thing, surviving 60% ice under her conditions is very different
from surviving 60% ice after cryogenic storage. On the other side, "survival"
by her criteria could easily be too strict a condition to warrant sacrifice
of other considerations. >>

Here we wholeheartedly agree.  "Smith's Criterion" was never intended as
anything *but* a starting place in the absence of concrete data.  Look at what
happened: as soon as we started getting feedback from histology and EMs we
changed our protocol.  I couldn't agree more that hamsters without
cryoprotection at -0.5C say little to what you get when you cool to -196C.  But
in those days it was all we had.  Sad to say, 15 years later I'm not sure we're
that much better off.



I would also suggest that the Ukranians do a head or two wherein cooling is at a
higher rate.  Say 24 hours from dry ice to liquid nitrogen temperatures.  It
would be especially useful to know if these brains crack.  If they don't, we've
eliminated the most likely variable (slow cooling to below Tg).

Mike Darwin

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