X-Message-Number: 4634
Date: 17 Jul 95 08:11:17 EDT
From: Mike Darwin <>
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 theabove 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 inthe -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


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