X-Message-Number: 2523
Date: 07 Jan 94 17:33:38 EST
From: Mike Darwin <>
Subject: SCI.CRYONICS Re: Perfusing Sheep Brains

Tim Freeman has asked some questions about the CI sheep head research and 
the apparently contradictory findings to the results obtained by Alcor and 
Biopreservation, Inc. (BPI).  I will try to answer his questions.

First, the short answer is that I cannot tell you what the differences 
were/are because CI has never published an appropriate, technically 
detailed description of what they did.  If you wish to know the results 
and the conditions under which the Alcor/Cryovita research was carried out 
please consult the draft of the paper "The effects of cryopreservation on 
the cat" by Darwin, et al.  This was posted on CryoNet some time ago.  I 
can repost it to a special reference file if you did not or cannot obtain 
a copy.  I hope to have this paper in print (with accompanying light and 
electron micrographs) in Thomas Donaldson's journal NEUROCRYOBIOLOGY 
(providing, of course it is accepted).

To BRIEFLY summarize our results: we found macroscopic cracking of the 
brain, spinal cord, and other organs, as well as poor ultrastructural 
preservation.  These results confirmed similar earlier results (with 
respect to fracturing) observed in humans.  The human cases were reported 
on in great detail including preparation method (i.e., perfusion cooling, 
etc.) and conditions under which rewarming and autopsy were carried out.  
The relevant cites for this information are as follows:

Federowicz, M.G., et al., Postmortem examinmation of three cryonic 
suspension patients. Cryonics 5:16;1984.

Leaf, J.D., Case Study KVM suspension. Cryonics August, 1981 pp 8-18.

Leaf, J.D., Case report: two consecutive suspensions, a comparative study 
in experimental suspended animation. Cryonics 6:11;1985.

Alcor Foundation, Histological study of a temporarily cryopreserved human. 
Cryonics #52, November, 1984 pp 13-31.

The protocol by which these patients (and by which animals) was treated 
consisted of perfusion of multimolar concentrations of glycerol followed 
by slow cooling to -196C (see papers for details).

Additionally, these same effects, i.e., fracturing upon cooling to below 
the glass transition point, have been observed with bulk solutions by 
several diffrent investigators over a long period of time starting with 
the paper by Kroener and Luyet:

Kroener, C., and Luyet, B.J., Formation of cracks during the vitrification 
of glycerol solutions and disappearance of the cracks during rewarming. 
Biodynamica 10:1966 pp. 47-52.

Rapatz, et al. also found cracking to occur in frog hearts cooled to below 
Tg after impregnation with multimolar concentrations of ethylene glycol.  
I do not have this cite at my fingertips but can dig it up if you REALLY 
want it.

Rubinsky et al, did a mathematical analysis of this phenomenon:

Thermal stresses in frozen organs. Cryobiology 17:1980 pp.66-73.

I also have a number of unpublished micrographs by Suda, et al. 
documenting cracking on a histological level in cat brains trated with 15 
(v/v) glycerol and frozen to -79C.  Suda also observed oozing of blood 
from the pial surface during reperfusion and even noted the presence of 
small visible cracks.

In personal communications Dr. Greg Fahy of the Red Cross Blood Research 
Lab in Bathesda, MD has also observed fracturing in organs (primarily the 
kidney and brain) treated with multimolar concentrations of cryoprotectant 
(glycerol, or DMSO-propylene glycol-formamide mixtures).  For this reason 
Dr. Fahy is pursuing cryopreservation through vitrification at slightly 
below Tg, not at liquid nitrogen temperatures.

Again, from personal communications with those in the tissue bank 
industry, I can report that viably preserved human heart valves will 
develop fractures (ruining them) if they are cooled to liquid nitrogen 
temperature.  For this reason valves are stored in liquid nitrogen vapor.  
I believe there have been published reports about this problem and I will 
do a literature search to see what I come up with.

As to what protocols are currently in use for human cryopreservation, I 
CRYOPRESERVATION which he can make available for downloading to interested 
parties (it is over 50K and so is too long for posting here). 

Now, as to SPECULATION as to why Ettinger and more recently BioTime 
researchers have not observed fractures.  Visible fracturing does not 
occur in my experience in brains poorly loaded with cryoprotectant.  In 
fact, about 8 years ago Jerry Leaf and I subjected neonatal lambs (which 
had been sacrified in another, unrelated study) to straight freezing to 
liquid nitrogen temperature.  We then tried cutting them up with various 
types of saws.  The purpose of this research was to develop a safe, 
reliable, and RAPID technique for converting already frozen whole-body 
patients to neuro (the need was urgent since three patients would have been 
otherwise thawed if this were not an option).  One of the things we 
noticed was the absence of macroscopic (i.e., visible) fractures in these 
animals even after sawing!

In our work with cats and rabbits we found that a general rule of thumb 
was that the higher the concentration of cryoprotectant (glass former) the 
more fractures.  Fractures do occur in straight frozen animals, but are 
smaller.  Anyone who has looked at an ice cube in a tray will understand 
about fracturing in ice, even at relatively high subzero temperatures.

So, one reason fracturing may not have occured is that the glycerol 
concentration in the tissue may not have been very high.  Again, without 
data on arterial vs. venous glycerol concentration as a minimum, it is 
hard to evaluate the results. On the other hand, CI observed shrinkage of 
the brains in response to glycerolization and that is a reasonably good 
indicator of gross adequacy of distribution of the agent.

Another reason fracturing may have been absent is the cutting of window 
(in the CI studies) in the skull combined with brain skrinkage.  Isolated 
brains freed from contact with a container during cooling CAN be cooled 
down to -196C without fracturing or with minimal fracturing.  This is 
particularly true if they are not handled in any way during the storage 
interval and are both cooled and rewarmed very slowly (G. Fahy, personal 
communication).  Again, we have no grapic or other data on the thermal 
protocols used by CI and thus have no way to determine what is going on.

One factor which I have come to understand contributes to the degree of 
fracturing is how much the system is disturbed mechanically after cooling.  
The cats, rabbits, and humans in which I have described fracturing were all 
moved (transferred from dewar to dewar or moved around in the dewar).  I 
believe this is a reasonable model since humans WILL be handled similarly 
over the time course of their storage and in fact have been so far.

Now as to the issue of using multimolar concentrations of agent.  Fahy and 
others have conducted studies of brains treated with 4M glycerol and 
frozen to dry ice temperature after which they were freeze substituted 
(i.e., ice removed by solvent and fixed at dry ice temperature).  The 
results were not encouraging: there was massive ice formation with cutting 
and disruption of the brain tissue at intervals of 10 to 30 microns.  The 
pictures are devastating, and speak for themselves.

Keep in mind that the lower your concentration of cryoprotectant the MORE 
ice you will see.  I have seen claims and heard claims from BioTime to the 
contrary, but have seen no credible evidence to support them.  I will 
believe these claims when I see EM and light microscopy documenting them.  
Reperfusion of brains with dye after freezing is easy and they look quite 
impressive.  I can easily get beautifully dye-filled brains (pial vessels) 
after glycerolzing, freezing to -79C, and thawing.  But that does NOT tell 
the tale of what is happening to the fine structure of the brain.  Of 
course, if I cool to -196C with the brain in the head, fracturing will occur 
and prevent good distribution of dye solution.

I have a projection slide from Suda showing cinematography of cat brains 
treated by various methods being reperfused with carbon black.  Brains 
treated with plain old glycerol did beautifully by this test.  But the 
histological injury was still there.


Responsible scientists working under well controlled conditions uniformly 
report gross mechanical injury from ice and fracturing using techniques 
broadly similar, and in some cases identical, to those in use by 
cryonicists today.  My own conclusion is that the degree of histological 
and ultrastructural preservation we are achieving is dismal using existing 
cryopreservation techniques.  We currently have a study underway using 
whole dogs perfused with ca. 7M glycerol which will look at a variety of 
parameters including the presence/absence of fractures during cooling to 
-135C or thereabouts.  There will be detailed documentation of the results 
of these studies published along with any claims made.

[ You can retrieve the BPI cryopreservation protocol, which Mike Darwin
  mention above, by sending email to me with the Subject line:
  "CRYOMSG 0028". - KQB ]

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