From: Mike Darwin <75120.575@compuserve.com>
Subject: CRYONICS Fixation & Rewarming
Date: 10 Aug 1995

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Date: 09 Aug 95 15:58:25 EDT
From: Mike Darwin <75120.575@compuserve.com>
Subject: CRYONICS Fixation & Rewarming

Joe Strout posted some suggestions and ideas about fixation and
subsequent cryopreservation.  There should *definitely* be a part in the
FAQ which deals with this issue because it comes up regularly and is
tedious to answer over and over again.

First, this is not a new idea.  It has been around for at least 15 years
and was the preferred scenario in Drexler's ENGINES OF CREATIONS.

Second, Joe is quite right about fixation making subsequent
cryoprotection problematic.  In fact, I'll bet Joe might actually have
fixed a few brains or animals by now.  Aside from protocols based on
fixative volumes needed vs. specimen size, a well known way to tell when
its time to stop perfusing with fixative is when the vascular resistance
begins to climb precipitously.  Fixation alters capillary structure and
function to essentially bring flow to a halt.

Third, any neurophysiologist or electron microscopist worth his/her salt
will look at you in horror if you propose fixing an animal via perfusion
after say, even 20-30 minutes of normothermic ischemia.  There are two
reasone for this: a) Many artifacts will appear (such as injured
mitochondria and clumped chromatin) which could cloud the issue of where
the injury came from (i.e.,the experimental procedure being evaluated, or
from ischemia).  This is not the major concern for cryonicists.  b)
Relatively modest period of ischemia result in very poor distribution of
the fixative.  Red cells become rigid when ATP depleted and stick in
capillaries, I-CAM is activated causing leukocyte and RBC plugging, and
regional cerebral blood flows go to hell through loss of normal
vasomotion.  Prolonged, low pressure cryoprotective perfusion seems to
open up many capillary beds which would remain closed during fixative
perfusion.

Of course, it could always be argued that fixation could FOLLOW
cryopreservation.  We have found this problematic in that the very high
viscosity of the perfusate inhibits fixation, as well as probably
cryoprotectant induced alteration of proteins in ways that make them less
likely to go through the condensation reaction wherein the hydroxyl and
amine groups on proteins react (by crosslinking and producing water as a
byproduct) and become "fixed."

Another point of great importance needs to be made here as well, and Joe
alludes to it.  Aldehyde fixatives fix proteins.  They do not fix
carbohydrates or lipids.  While carbohydrates may not be very important
to memory, identity, and so on from an information-theoretic standpoint,
it would be hard to argue that lipids are equally inconsequential since
they comprise the major load of the membrane structure which is both the
identifying border of cells and the locus for many, many critical
biochemical reactions which undoubtedly are important to identity.

In order to fix lipids you must generally use a metal like osmium in a
reactive form (such as the tetraoxide).  Osmium is not, I repeat not,
nice stuff.  You might be able to substitute mercury compounds or even
arsenicals, but the resulting mass of tissue would be not only mildly
poisonous, as is the case after aldehyde fixation, but highly poisonous
and have a top-drawer haz mat classification.  In fact, the use of heavy
metals for embalming corpses is specfically forbidden by law in many
states (maybe all?) in the US for two reasons: danger to the ground water
and environment and, more appropriately, because salts of metals like
arsenic and mercury make such good poisons for the purpose of rendering
otherwise healthy (or at least not immediately terminal) people dead.
This is called homicide.

A major reason for the deterioration Joe spoke of in fixed materials is
that MOST of the specimin is not fixed.  Anyone who has stored a human or
animal brain in a jar of aldehyde fixative will observe the progressive
leaching of compounds into the bathing media and the progressive
deterioration of the tissue macroscopically over a period of several
years.  Indeed, in specimins where the fixative level was allowed to drop
exposing the specimin in a closed jar, I have observed a white, fuzzy
mold growing quite nicely on such tissues -- presumably in a formaldehyde
saturated atmosphere!

Finally, what no one in this discussion has considered is the effect of
fixation on cryoinjury.  My expectation was that it would reduce it since
everything should be turned into a rubbery polymer.  Certainly cells in
culture which are fixed and repeatedly frozen don't dissolve away like
unfixed freeze-cycled controls.

However, one nasty consequence of fixation is that it reorganizes
membrane structure and opens large pores in the plasmalemma.  These pores
allow free movement of water, cryoprotectants, salts, and even fairly
large molecules like mannitol (MW ca. 190).  As a consequence, cells do
not dehydrate when freezing occurs and ice propagates right across the
cell membrane resulting in intracellular freezing.  EM examination of
such tissue is a dismal business.  The tissue looks WORSE than if it had
been straight-frozen without any cryoprotectant or fixative at all.

Finally, as alluded to earlier, fixation requires the *diffusion* of
chemicals and for diffusion to be effective and rapid enough to inhibit
autolysis the maximum distance from fixative to tissue in need of
fixation can be no greater than 1 mm and preferably no more than 0.5 mm.
In many, maybe even most of today's patients, you are not going to get
good distribution of fixative, period.  Cooling, on the other hand, while
slow in large masses, still will eventually reach ALL of the tissue and
in a far more timely manner than fixation.  Its effects are thus
independent of the condition of the circulatory system or distances
between the surface and core of an un- or underperfused area.

As to heating, I see no need for little pellets full of nasty, highly
reactive compounds. Existing RF technology can rapidly and uniformly
rewarm masses the size of human kidneys in excess of 300 to 400xC per
minute.  Theoretical consideration and some clever design work have
demonstrated that the limits of RF heating are not particularly mass
dependent -- at least not as far as a human body is concerned.  Whole
humans can, in theory at least, be rewarmed at comparable rates even with
the very limited consideration given to this problem so far.  Adequate
cryoprotection, present either before or after repair would make this
problem much easier by eliminating the presence of ice.  Such
cryoprotectants would also serve to greatly relax the requirement for
very high rates of rewarming to avoid ice formation after repair, from
thousands of degreees C per minute to hundres of degrees -- or even
lower.

I find I must agree with Charles; some of these scenarios, given the
technological capabilites they posit, certainly are Rube Goldberg ways of
achieving what might simply be done by other means such as using the same
end-stage nanotechnology in combination with simple, macro approaches
such as RF rewarming.

Ralph's scenario and the background which produced it remind me of the
old adage: When all you have is a hammer, every problem looks like a
nail.

Mike Darwin

From: Mike Darwin <75120.575@compuserve.com>
Subject: CRYONICS:Request for help
Date: 15 Oct 1995

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Date: 14 Oct 95 23:54:58 EDT
From: Mike Darwin <75120.575@compuserve.com>
Subject: CRYONICS:Request for help

REQUEST FOR ADVICE/HELP:

I have always said I would take help where I can find it.  Further, I have
always said I'll be the first to admit I am in 'No man's land." when I am
where patient well being is concerned, and cry out in the wilderness.

BRIEF CASE HISTORY:

A 32 year old caucasian female in previous good health experienced sudden
cardiac death intraoperatively due to iatrogenic causes.  She was rmoved to
the ME's office within one hour of arrest, air cooled for 24 hours at 4 C
and the subjected to autopsy.  The brain was removed very nicely en block
(best job I've ever seen) and hung in a pail of formalin by her vertebral
arteries for about 30 hours at 4 C (as far  as I can tell the formalin was
room temperature when the brain was placed in it, and the the brain in the
plastic bucket was put in morgue cold room).

The brain was NOT sectioned.

The husband contacted a cryonics organization which allegedly advised him
to remove the brain from formalin, rinse it with saline and freeze it to
dry ice ASAP.  As is not uncommon in such cases these instruction got
garbled up (we ALWAYS use FAX for follow up on any verbal info given in
cases like this). The husband had the brain REMOVED from formalin and put
in saline solution at 4 C for about 4 weeks.

The brain arrived courriered by the husband and Mark Connaughton of
CryoSpan, cooled to 2 C on ice at our facility and had been returned to
formalin (per our instructions) the day before. On opening the container
the brain was found in a shallow pool of formalin containg liquiud and was
covered in a formalin soaked muslin surgical towel. On palpation the brain
was found to be very stiff and uniformly hard, no "soft" areas could be
detected in the interior by gentle palpation.  The visual impression was
one of old fixation (charcoal black vessels on a pearl white background; no
sign of undenatured hemoglobin present).

The brain was then transferred to a perfusate base called SHP-1 which
contains buffer and sucrose, instead of mannitol (no problem with sucrose
toxicity here, no need for mannitol's pharmacologic effects in mitigating
ischemic injury and brain edema during CPA perfusion) due to sucrose's
better glass forming properties.  A marker non toxic dye of a desireable
molecular weight was also present in the continuously stirred 13 liters of
fixative solution (no colloid present).  The brain was suspended above a
stir bar in the turbulent fixative solution in an open mesh nylon laundry
bag of the kind used to launder socks and small items that might otherwise
be mixed up and made difficult to sort, or go down the drain.

Samples were taken for light and TEM.

After 24 hours in the fomalin the brain was transferred to the same,
precooled solution plus 2% glutaraldehyde ("glute").

THE PROBLEM:  Fixing opens cell membranes and allows formation of
intracellular ice, preliminary work by Fahy (unpublished) has shown that
freezing damage is greatly exacerbated in fixed tissues, especially badly
in straight freezing, and not much better in the presence of modest levels
of cryoprotectant where any significant amount of ice is formed.

We have prior experience with glycerol diffusion times, and it takes nearly
a year to reach equilibrium (we hope) with a 72% concentration.  For many
reasons we do not wish to repeat this procedure.

We are thus confronted with a "black-box" conundrum; since it has already
been four weeks (through no fault of our own) and since the brain is
already quite rubberly and appears well fixed (again through no fault of
our own) (Yes, we are aware of the limitations of formalin fixation and are
adding glute to the system for this reason).

We can either go ahead and freeze her, try to cryoprotect her, try to load
enough CPA into the likely well fixed (and presumably most important) gray
matter, or try to go for broke and load the whole brain with a vitrifiable
amount of CPA.

Our previous studies with glutaraldehyde/formalin immersed dog brains had
shown poor fixation of the interior even at 5 days at 1-2 C.  However, more
recent work with formalin alone has shown full thikness penertration of
fixative in *dog* brains placed in room  temperature fixative and
refrigerated to 4 C over night.

Our current plans are as follows:

1) We have settled (so far) on using a modifed VS-4 Solution consisting of
DMSO, propylene glycol and ethylene glycol (EG).  We have avoided using
amine containing compounds like formamide and acetamide (the normal
ingredients in VS4 which balance the DMSO and PG) because they will cross
react with formalin and glute and complicate repair by forming all kinds of
bonds and structures with native fixed protein and then, undego
conformational changes and other reactions which will make inference or
repair more difficult. We have substituted the formamide mole per mole with
EG (mol. wt. of  approx. 64) to yeild a final v/v concentration of CPA of
about 69%.

2) We have toyed with the idea of using glute and formalin in very high
concentrations as glass forming adjuncts, replacing DMSO, or perhaps other
elements in whole or in part in VS4.

3) We have also considered using methanol and methanol and other CPA
mixtures.  This is very attractive because of methanol's rapid penetration.
 But, it has not been chosen yet because of methanol's excellent ability to
act as a lipid solvent and to dissolve some fairly impressive cross linked
materials: it dissolves polymerized coatings (paints) and many plastics, and
it used routinely as an industrial solvent for degreasing, dissolving
lipids, etc. DMSO is too, but truly there is no comparison.

We are concerned here about methanol's lipid solubilizing effects because
the lipids may be be critical in allowing "reconstruction" of the patient.
For instance, even in human brains at 24 hours of ischemia intermediate
between 37 C and 17 C (i.e. air cooled to 17 C) we see myelin (granted,
unravelled and frayed) without axoplasm, but the path of the myelin can be
followed on light and TEM (serial sections) and on the light level we can
see cell membranes (neurons mostly, glial cells give up the ghost early).

We are thus reluctant to risk eluting any material from the system.  We are
clearly in this case in the classic position of being between Cylla and
Caribdis.  More DETAILED information on methanol fixation would be useful
(I have a file on this that is about 10 years old).  In particular, if
Skrecky has papers at hand (rather than just abstracts, although I'll take
them too) I would like to get my hands on them quickly.

Extensive prior experience with osmium, and other reactive metal-lipid
fixing agents has shown a total failure to penetrate beyond a few mm of
tissue even after a year of exposure (this work has been confirmed in other
labs as well).

The circulatory system is completely inaccessible.

QUESTION: Does anyone have any reasonable suggestions here in terms of the
CPA mixture?

(Doug S., save your time about sucrose, we have 55 g/l present now and it
diffuses horribly slowly. Also, forget about needles to give CPA, there are
a dozen reasons why this isn't an idea for consideration in this case.
Ditto sliceing up the brain: I can barely get the husband to let me do
needle biopsies to track dye penetration, plus I don't think the salami
technique a good idea myself).

QUESTION: We want to see if whatever we do is working in terms of CPA
penetration.  One idea I have had is to add Hypaque, an iodinated dye used
in angiography, to the fixative/CPA bath in order to track noninvasively
the penetration of CPA with CT.  Unfortunately, it has a MW of about 800.
I got the following from Brian Wowk in response to a query for other
radiopaque markers:

>As you suggested, the Merck Index was a rich source of iodonated
>x-ray contrast agents.  They are all basically benzene rings with iodine
>atoms and various organic compounds attatched to the corners.
>Unfortunately the water-soluable intravenous agents used clinically
>are all around 800 Daltons (glycerol is around 90, I think).  There
>is another class of presumably fat soluable agents used for
>cholecystography that is around the 600 range, but fat soluble agents
>are obviously useless to us.

>The only possible hope I saw was something called Iopydol
>1-(2,3-Dihydroxypropyl)-3,5-diiodo-4(1H)-pyridinone that weighed
>in at 420.99  No indication as to solubility is given, but with a
>couple of OH groups hanging off the organic group, it *might* be
>water soluble.  It used to be used as an inhaled (aerosolized?)
>agent for bronchography, but since CT was invented, I don't know
>if anyone still does bronchography, or where you could get this
>stuff.

>Still think your best bet is T1-weighted MRI to get an
>at least qualitative idea of where the CPA has gone.
>For the case you currently have on hand, your
>dye idea doesn't sound too bad either.

The ideal marker MW would be no lower than 60 and no higher than 150
daltons.

This would allow us to do CT repeatedly which is much cheaper than MRI.  We
have an "imaging" budget of $800 on this case.  We will probably cool the
brain as fast as we can to the Tg of the CPA mix we use once loading is
complete.  If we have evidence of thorough penetration we may cool more
slowly as the end concentration of non water agents in the system
(including the 5% v/v of fixatives) will be over 70% (a concentration which
should be stable during moderate rates of coling and which will not allow
propagation of ice).

 If we see very poor penetration of CPA we are in a quandry because while
we can cool the outer layers of the cortex reasonably rapidly (and these
are presumably the best preserved areas since they were fixed first) if
there is a lot of water inside the brain which will expand upon freezeing
or have a different coefficient of contraction on cooling past Tg than the
high concentration CPA loaded outer layer of the organ, we may see
extensive shell fracturing or stress fracturing of the cortex.

Suggestions anyone?

ETHICAL CONSIDERATIONS

The husband is a highly intelligent professional with solid finances. He
has long been interested in cryonics and life extension and has read
Nanosystems.  He and his wife were to retire and move out of state soon,
(both are in their 30's) and they had planned to keep their eye on cryonics
groups and "make arrangements" closer to their time of need/risk (50's)
after following the various groups over a long baseline. Both were in
excellent health.

I personally have spent many hours on the phone with this man, and he has
an excellent grasp of how dismal the situation is.  His final words on the
subject were (paraphrasing): "Well, there may not be much structure there,
but whatever IS there will be gone for good if I do nothing.  I can afford
this, she would have wanted it, and I see no reason not to do it."  He has
been actively involved in selecting the cryopreservation protocol being
used, and will be involved in evaluating suggestions from this forum and
any others you may forward it to.

My professional opinion is that the *best* that can be said about this
patient's prospects for recovery with declarative memory/former identity
intact are that they are vanishingly small.  I and others from other
cryonics groups have repeatedly informed the husband of this and explained
the biology of autolysis, synapse degradation (dendritic spine loss), and
the likely heavy added injury from ice if we cannot diffuse in enough CPA
to vitrify any non-autolyzed remaining structure.

The husband is four weeks out from the loss.  He is calm, rational and
composed and has had time to reflect on his decision.  He is being given an
opt-out period of some months to change his mind.

MOST DESIRED CONSULTANTS:

Input from Joe Strout and others involved in investigating the neurobiology
of memory and learning as active researchers are especially welcomed.  If
we could better guess what we can likely SAFELY "afford to throw away"
(i.e., dissolve or elute) that would be useful information.

Input on alternative markers for imaging are also solicited (we have some
experiernce with D2O doping, and may use this too, cost permitting, but its
MW is too low to be highly desirable).

PERSONAL:

Based on currently available information this woman died from (in the ME's
opinion, and mine and that of my medical consultants) of iatrogenic causes
(in other words medical error).  She left behind a very young child.  She
was an extraordinarily beautiful woman physically and, from what I've seen
(her husband had her wallet/purse with him) she was a gentle, loving mother
to her child she had pictures of her child and fortune cookie fortune
lovingly attached to one of the child's photos..

It is apparent that her husband loves her very much.

Any help would be appreciated.

If you wish to speak with me by phone please send me your phone number(s)
via email and a contact time.

Thank You,

Mike Darwin, President
BioPreservation

> Date: 15 Oct 95 00:23:47 EDT
> From: Mike Darwin <75120.575@compuserve.com>
> Subject: CRYONICS: addendum to help

BPI has requested help on a case in process from Cryonet subscribers.
Unfortunately, I forgot to add two important pieces of information:

1) You are welcome to cross-post the request for help to any reasonable
lists for the purpose of eliciting REAL help.

2) DO NOT cross post, forward, copy other lists without checking with me
BEFORE doing so.  My purpose here is to avoid certain likely target lists
from receiving the SAME forwarded message from 20 people.  Let's coordinate
who gets this so that there in chain-reaction propagation.

3) The partient's current status is suspended in fixative bath (well
stirred) at 3.2 C in an open mesh Nylon bag.  Fixative contains 3%
formaldehyde, 2% glutaraldehyde, sucrose, electrolytes and buffers.  No
cryoprotectant has been added yet (we are in essence post-fixing with glute
while we decide what to do next).

Mike Darwin