From: Mike Darwin <75120.575@compuserve.com>
Subject: SCI.CRYONICS Re: clarifications
Date: 20 Jul 1995

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Date: 17 Jul 95 08:11:17 EDT
From: Mike Darwin <75120.575@compuserve.com>
Subject: SCI.CRYONICS Re: clarifications

I want to thank Bob Ettinger for his posted corrections to THE IMMORTALIST
article which I sent him e-mail regarding.

I also want to widen the scope of the discussion a little, and to comment
specifically on the issues of toxicity of glycerol as it relates to the BPI
and CI methods.  Some of these issues I've raised with Bob privately.
However, after my last message to him on this subject I realized that I had
forgotten to include some crucial analysis.

I am home now, and do not have easy access to my hard-drive at the lab. (I
apparently just managed (through an act of willful stupidity and
impatience) to refracture my metatarsal at the site of the old break less
than 10 days after having a cast removed: X-rays tomorrow will telk the
tale.)  Thus, I will be relying on memory about some of the discussion that
passed between Bob and I, and I trust that Bob will both correct and
forgive me if I state his positions wrongly.

In his IMMORTALIST article Bob stated (paraphrasing) that BPI (and Alcor)
have made a trade-off using higher glycerol concentrations in exchange for
less ultrastructural injury.  This is, in a narrow sense, is true. But
first some background.

Pegg and his associates in England hold, I believe, the record for
introducing and removing successfully the highest concentration of glycerol
in a mammalian organ.  They used the rabbit kidney as the model and
transplantation of the glyced and deglyced kidney was followed by
contralateral nephrectomy (delayed, if I recall correctly) so that the
glyced-deglyced kidney supported the animal as the sole kidney.  Again,
being handicapped by being away from my back-issues of CRYOBIOLOGY as well,
I must recall from memory the details of that work.  As I recall, the
maximum concentration they were able to reach without subsequent necrosis
of the kidney or injury sufficient to prevent the autotransplanted kidney
to support the animal as the sole kidney was about 3M glycerol. Even then
the concentration of waste products rose in theanimals blood and remained
elevated (BUN and creatinine).

It is very important to point out here that these kidneys *were not*, I
repeat, were NOT frozen.  They were perfused to 3M glycerol and then
deglyced and reimplanted into the animal from which they were originally
removed.

Pegg found that the kidneys were exquisitely sensitive to how glycerol was
both introduced and removed, and further (if I recall correctly) that the
ionic composition iof the carrier solution (base perfusate) was also
critical to success.  Failure to use the "right" carrier solution resulted
in nonviable kidneys in the presence of 3M glycerol. (It is quite possible
that composition of the base perfusate was important in moderating glycerol
toxicity in some way.)

Many similar studies with other osmotically active (i.e., colligative)
cryoprotectants in solid mammalian organs have confirmed the following
general principles:

1) Rate of introduction and removal are critical to avoid osmotic injury
both on the cellular level and at the tissue level.

2) Cryoprotectant toxicity for a wide range of agents is related to a
limited, but significant extent, upon the amount of water replaced in the
system by the cryoprotective drug.

3) Temperature of introduction is critical in moderating toxicity, but also
in facilitating or inhibiting cellular permeability of the agents.  This is
particularly true of glycerol where there are known interspecies
differences in the temperature-tissue equilibration profile.

The technique used by Alcor during my tenure there, and by BPI now, seeks
to try, within far more generous limits than those found necessary by Pegg
and others, to follow the path made by other solid organ cryoprotective
perfusionists by gradually introducing glycerol and progressively reducing
the temperature.  For instance the maximum cryoprotective agent (CPA)
concentration (in this case glycerol)  the patient is subjected to at the
start of perfusion is 5% v/v (unless long ischemic times have occurred in
which patients 10% v/v glycerol may be used at the start.)  Subsequent
increases in CPA concentration are achieved by adding base perfusate
containing very high concentrations of glycerol to a mixing reservoir in a
closed loop with the patient's circulation, while at the same time removing
a comparable (or at leasted controlled) volume of perfusate from venous leg
of the same recirculating system and dumping it down the drain.  This
results in a more or less linear increase in glycerol concentration.  We
have reason to believe that a linear approach is probably not the best, and
that more accelerated rates of addition (with concomitant lowering of the
temperature) may be appropriate as the tissue is progressively loaded with
CPA.

Certainly, Greg Fahy has demonstrated this with Vitrification Solution 4
(VS-4) (a mixture of DMSO, propylene glycol, formamide and a colloid):
perfusions starts above 0 C with relatively modest rates of introduction
while the temperature is progressively dropped.  A faster rate of
introduction is then used for final loading, and perfusion is concluded at
-20 C or thereabouts (yes, that is correct MINUS 20 C) with total
concentration of VS4 near 50% v/v..

CIs approach is different. As I understand it, CI patients are perfused
with 75% glycerol solution in one step using an open circuit approach.  I
do not know the volume of perfusate used, but I have been informed by Bob
Ettinger that terminal glycerol concentrations in the venous effluent are
in the vicinity of 25% v/v (or near 3M).

It is my interpretation that Bob is of the opinion that this approach
obviates the toxicity of high concentrations of glycerol or at least
minimizes it compared to BPI's approach.

In my private communications with Bob I pointed out that if we are to play
the viability game, we must play by all the rules and not just go by the
final numbers.  Introduction of 75% glycerol straight-away will, I
guarantee you, render tissue nonviable in most organ systems by current
criteria.

Additionally, while the mixed venous return effluent may contain only 25%
or so glycerol, it is very important to understand that in the ischemic
brain (even after comparatively brief periods of ischemia) and especially
in the profoundly ischemic brain (typical CI patient, and all too typical
of cryonics patients in general, regardless of which organization or
company they have been treated by) regional brain flow becomes radically
altered and some areas of the brain will receive far more perfusion than
others, no doubt resulting in some areas receiving very high concentrations
of CPA and other receiving very little.  Shortly before his
cryopreservation, Jerry Leaf was on his way to demonstrating this in cats
using radioactive microspheres to assess regional flows.

Further, the afferent end of the capillaries receiving 75% glycerol will
reach a far higher concentration than the efferent end.  Also, normal
autoregulation of flow will be absent in profound hypothermia with marked
shunting going on.  We are rapidly coming to the conclusion (as are some
clinical investigators) that some brain injury to asanguineously perfused
dogs in deep hypothermia (a survival model) is due to shunting: in other
words the normal opening and closing of the precapillary sphincter is
absent or deranged and flow takes the path of least resistance, leaving
some areas  of the brain under-perfused, or not perfused at all.

These were points I made to Bob in our recent communications, although with
more brevity.

However, a point I did not make is that even assuming the CI technique
yeilds a uniform (and "safe" i.e., not toxic to kidneys) 25% concentration
in the tissues with no high concentratiion areas, what follows makes this
irrelevant.

During my tenure at Alcor and at BPI, after loading the patient with CPA we
cool him or her as rapidly to a little ways below the freezing point of the
7.4M glycerol as we can and, once freezing is over, we march the
temperature at a minimum of a 10 C surface to core differential to -79 C.

CI, by contrast, places the patient after perfusion in a sleeping bag and
then gas cools the patient to -79 C using dry ice over a period of 1-week.

This process (Ci's) of cooling is not linear and has long concerned me.
Dating back to my earliest days in cryonics I am intimately familiar with
how much any insulation on the patient slows cooling.  Even double bagging
the patient in plastic bags (from which air is evacuated with a shop-vac)
greatly slows cooling when the patient is transferred from the OR table at
a few degrees above 0 C to a -40 C bath.

A patient in a sleeping bag with passive (non stirred cooling) will not
only cool ver slowly, but will spend a lot of time at the freezing point of
the glycerol-water solution in his/her tissues.  This is so because the
latent heat of fusion must be dissipated.  And, for a patient with only 25%
of the body water replaced with antifreeze this represents a substatial
amount of heat (about 80 calories per gram of water).  Add to this the
relative poverty of gas cooling and the presence of significant insulation
and the following scenario emerges:

1) Cooling to the freezing point of a 25% glycerol solution will be very
slow (data from the Berkowitz case where small amounts of fiberglass
insulation were used to buffer dry ice cooling will bear out this fact).

2) Once the freezing point of the 25% glycerol solution is reached (ca. -4
C), a great deal of time will spent there while water is converted into ice
and heat dissipated.

3) Following freezing, the rate of temperature descent will increase
markedly, but will still be slowed by insulation (sleeping bag) and by the
decreasing spread of patient to air bath temperature.

There are, in our recent experience (and past theoretical concerns) several
corollaries that flow from the above considerations:

1) Patients treated in this way will be exposed to glycerol for
(comparatively) long periods of time at relatively high temperatures before
freezing begins.

2) Once freezing begins it will take a long time to complete due to the
slow rates of heat exchange (insulation, air cooling, minimal convection,
etc).  The downside to this is that, as freezing proceeds, the glycerol
concentration will rise steadily and to very high levels.  Unfortunately,
this large increase in glycerol concentration will be taking place at very
high subzero temperatures over a long time course.

This is one of the reasons why I have pressed Bob and CI so hard on
providing actual patient cooling curve data.  From such data it is possible
to see the isotherm (freezing point of the tissues), to see how long the
tissues remain at the isotherm, and thus to determine indirectly but fairly
accurately:

1) What the average glycerol concentration was in the tissues during
freezing.

2) How long the patient was exposed to a given concentration of glycerol
during freezing.

This is one of the reasons Jerry Leaf and I began placing a temperatures
probe on the brain surface following perfusion: so that we could see how
well we did with glycerolization and get some idea of how long the patient
was exposed to the eutectic (or intermediate) concentrations of glycerol
during freezing.  BPI plans to expand on this idea by placing carefully
pre-calibrated probes in many areas of the brain not only during subzero
cooling, but also  during perfusion: such probes provide valuable insight
(indirectly) as to the regional flows of the brain.  Modest, deliberate
adjustment of the temperature of the perfusate (and maintainence of a
constant external temp.) allow a sort of thermal map of flow to be made and
imaged by computer.

So, what is my point here?  Simple really. The fact that CI's terminal
efflluent glycerol concentration is only 25% in all probability has little
to do with toxicological injury to the patient from glycerol.  Further, it
is not only possible, it is probable that CI patients are exposed to far
higher concentrations of glycerol at far higher temperatures for much
longer periods of time than are BPI and, presumably, Alcor patients.

We now have some strong indirect data on this (from rewarming experiments)
with direct data on the way (from cooling/rewarming) experiments:

When we first began thawing our glyced dogs (7.4M) we used the Suda
technique of allowing them to thaw in a refrigerator set to near 0 C with
the animals wrapped in a warm weather children's sleeping bag.  Rewarming
took over 48 hours with most of the time  being spent at around -12 to -16
C in the presence of near eutectic concentrations of glycerol (i.e., in
excess of 8M).  The first thing we noticed at necropsy prior to fixative
reperfusion was the "off color" of the tissues.  The tissue didn't look
right (darker, abnormal colors with red staining) and upon reperfusion the
K+ was very high (over 40 mEq/L) and myoglobin was observed in large
quantites in the effluent.  In addition, no intact red cells could be found
in examining the effluent: only RBC "ghosts" were present, i.e., red cells
that had leaked out their hemoglobin.

The ultrastructure from these two very slowly thawed dogs was very bad.

Subsequently, we decided to very rapidly (by our standards) rewarm dogs
from -90 C by transferring them directly (and without a plastic bag) to a
cooling bath which was chilled to only 0 C.  Rewarming rate was on average
about 10 C per hour as opposed to 1-2 C/hour for refrigerator warmed
animals.  More to the point, the time spent near the eutectic point was
greatly reduced.

There was an immediate and marked difference in the appearence of the
viscera of these dogs both grossly and ultrastructurally.

Our next experiment (first half, cooling) completed last weekend will
attempt to further improve onthese results by increasing our cooling rate
by directly submerging the dog in the -40 C bath without plastic bag
protection.  Based on slice studies we expect these results to be even
better than was achieved with high rates of rewarming.

The point of this very long and very technical post is that things are not
simple:

1) Despite CI's lower final concentration of glycerol at the conclusionof
perfusion, the actual exposure time and temperature profile of CI patients
to very high concentrations of glycerol is likely to have been far longer
than for BPI patients, or others using liquid bath cooling with large
surface to core delta Ts..

2) We are all in the same boat in terms of high glycerol concentration
exposure of our patients at relatively high temperatures.  Incidentally,
this is why freezing rate is so critically important to survival of cells
frozen with CPAs.  As the ice forms it freezes out as PURE water and this
causes a rapid rise in cryoprotectant concentration.  Reduced temperatures
mean reduced toxicity.  Thus, cooling rate must be adjusted so that as
eutectic concentration of CPA takes place, the tissue is always "bailed
out" or protected from toxicity by the constant lowering of the
temperature.  For most cells a cooling rate of 1 C/min. is optimum.
Obviously, for large organs or whole bodies this rate of cooling is not
achieveable: not even by plunging the person into liquid nitrogen,
perfusing them with cold gas or liquid, etc.  There is just too much heat
to move.

Larger cells or small groups of cells stuck together and covered by a
membrane of course will lose water to surrounding ice more slowly (with
resultant slower rise in CPA concenrtration) and thus require different
cooling rates.  Embryos for instance do not like to be cooled 1 C/min. as
they end up freezing intracellularly and are thus killed: the embryonic
cells are surrounded by a gelatious barrier called the zona pellucida which
greatly slows water loss to extracellular ice during freezing.

When I was at Alcor and more recently at BPI I examined low concentration
approaches to brain cryopreservation.  Damage was far worse, as can be seen
the EMs published in CryoCare report.

We are all paying an unknown price in toxicity (hopefully confined to
noncritical biochemical derangements).  This does not make me happy, and,
even having paid such a price, we are still experiencing a lot of
cryoinjury.

Anyone who thinks we are "home free" yet or that we are safe is being
foolish.  And I would urge all of you to read Thomas Donaldson's message
about Ralph Merkle's Web page, or look at the interviews with Eric Drexler
in the last issue of EXTROPY magazine to get an idea of how optimism can
become a cancer eating away at incentive and corroding convergence on good
preservation techniques which, if not perfect, are at least far more
rigiorous, than our current, largely theoretical hit and miss approach.
While it is certainly true that just because ultrastructre isn't there when
we look for it with today's techniques it may be found by tomorrow's.  It
is equally true that it might not be found at all.  I think Donaldson is
right on the money in his criticiams of Merkle and deserves very careful
attention.

And besides, who in their right mind, given the choice, would want to rely
on theoretical hand-waving when they can have biological certainty or
something approaching it: and at modest cost and over reasonable
contemporary human time scales.

As I have said before, it is better to believe by SEEING than by faith or
clever tales spun about what *might* be.  Or, as my Mother often says: a
bird in the hand is worth two in the bush.  Ever chased a bird in brush?  I
have.  Mom was right.

Good luck.


Mike Darwin

From: Mike Darwin <75120.575@compuserve.com>
Subject: Glycerol
Date: 22 Dec 1995

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Date: 21 Dec 95 17:32:20 EST
From: Mike Darwin <75120.575@compuserve.com>
Subject: SCI.CRYONICS Glycerol

Doug Skrecky writes:

>Cryonicists use glycerol primarily because cryobiologists do.

Doug, this is just plain wrong.  And I think you know it, or should.  Here
are the relevant and *correct* facts:

1) Cryonicists did not always use glycerol.  We tried Me2SO (DMSO) early on
because of its greater cellular permeability and the superior protection
its provides some tissues (over glycerol).  This was a disaster with
patients becoming edemetaous (swelling) and circulation grinding to a halt
before significant concentrations of agent could be loaded into the tissue.
 The last patient I perfused with DMSO gained 30% of his weight in fluid
and ended up with an aortic DMSO concentration of 5% on autopsy (when he
was converted from whole body to neuro).

2) Extensive screening studies have been carried out using rabbit brain
slices and in some cases rabbit and dog brain perfusion preparations by
Fahy and by Darwin (me) independantly, using a wide variety of agents
followed by freezing and thawing.  Some of the sgents were:

DMSO (perfusion and slice incubation)
propylene glycol (PG) (perusion and slice incubation)
PG and DMSO (slice incubation)
ethylene glycol
glycerol and sucrose (perfusion and slice incubation)
glycerol and DMSO (slice incubation)
2,3 butanediol (slice incubation)
DMSO to 60% (v/v) using the Farrant technique of stepwise increase of
concentration while stepwise reducing the temperature to minimize toxicity,
using brain slices).
glycerol and trehalose
Vitrification Solution 1 (VS1) (perfusion of intact dog heads)

Not only were the above agents screened, but they were often evalauated at
different concentrations and with different temperatures of introduction
and different cooling/warming profiles. Results were evaluated by light
microscopy in all cases (often with two or more stains) and by EM in many
cases.  Freeze substitution was used in the most promising cases based on
post-thaw light and EM results.

EG gave comparable results to glycerol.  Most others were worse.  Some
looked OK at the light level, but caused serious ultrastructural disruption
at EM; like dissolution of synaptic membranes!

3) Glycerol-PG perfusate was, I have heard rumored, used on at least 2
early CI patients and resulted in massive edema.

4) I have been very unhappy with glycerol for years.  If you start at the
beginning of CRYONICS magazine and read all of them you will find this
unhappiness reflected in many remarks made over the years.  I am unhappy
with glycerol because:

a) It equilibrates poorly with brain, skin and skeletal muscle cells.  This
results in massive dehydration of these organ systems.  In fact, many
patients are positively mummified in appearance after glycerolization even
to 3M.  I mean this literally; a casual observer (excluding skin color from
consideration) would assume he was looking at a mummy; there is massive
water loss in some tissues; up to 50% for muscle and skin and slightly
under or over that for brain.

b) Glycerol does not penetrate myelinated axons well and intra-axonal ice
formation is massive.  I have distributed freeze-substitution pix (EM) to
Merkle, Donaldson and Ettinger which show a clear and easily demarcated
line between gray and whit matter in canine brain treated with 4M glycerol.
 The line of demarkaction is where the size and number of crystals abruptly
change  from gray to white matter.  A child of 6 could recognize and point
out the pattern difference and draw the line between the tissues with a
highlight marker (and yes, I've DONE this experiment using a visting child
to the lab).

c) The above notwithstanding, glycerol does equilibrate some, and it does
provide better protection than the above agents.

d) We are and have been for 6 months looking at sorbitol and several other
compounds.

e) I remarked to Keith Henson at the ACS Directors meeting held at the 21st
facility a few weeks ago that I sincerely hoped that I had perfused the
last *optimally* stabilized BPI cryopatient with glycerol.  This should
tell you that some of our new screening work has suggested a better
compound(s) for use in brains.  My remarks to Keith preceded your comments
here.

This new technique will possibly and maybe even probably not be applicable
to patients with long unstabilized down-times or capillary/extracellular
matrix injury because of the liklihood of such patients developing
perfusion-limiting edema; in such cases we will use glycerol.

f) Years ago, long before your involvement with cryonics, I switched from
mannitol in our base perfusate to sucrose because sucrose provided superior
membrane protection to synaptosomes and was a better glass former (based on
the literature).  We pumped three live dogs with sucrose base perfusate and
had three surviors  (2 hours of perfusion at 4 C).  However, we
subsequently had serious problems with dogs using sucrose (they died!) and
more to the point, we had markedly worse cerebral edema during
cryoprotective perfusion in humans; the more pre CPA perfusion ischemic
injury, the mor rapid and worse the brain swelling during glycerol-sucrose
perfusion.  Bob Ettinger observed the same thing with his sheep heads.
Despite mannitol's absent glass forming ability and its proclivity to form
large crystals on cooling, it still provided superior protection against
edema, and superior EM results, despite the obvious theoretical advantages
of sucrose.

The switch to sucrose is also documented in CRYONICS.  I suggest you get
the back issues on diskette and READ THEM (Steve B., for God's sake, for
all our sakes!, send Doug the back issues free; I'll pay for the diskettes
costs and shipping!) so we are spared his uninformed posts and/or can flame
him into silence or general disregard, with justification.

Finally Doug, we are not stupid and your native intelligence is great
enough that you do not have to be.  You do sometimes come up with useful
information; but it is like regurgitation on a plate instead of well
prepared cusine.  Sometimes nourishing, but very unpalatable.  Please learn
that science is not done in an arm chair; at least not since Plato and IMHO
that wasn't science.

Mike Darwin