X-Message-Number: 12595
Date: Tue, 19 Oct 1999 19:07:30 -0400
From: Mike Darwin <>
Subject: Sorry, no "major breakthrough" today

Doug Skrecky writes of the following paper as being a "major breakthrough"
in ameliorating cryoprotectant toxicity:

Effects of Electrolyte Composition and pH on the Structure and Function of
Smooth Muscle Cooled to -79 C in Unfrozen Media
Cryobiology 9: 82-100
B.C. Elford and C.A. Walter
Clinical Research Center, Harrow, Middlesex, HA1 3UJ

Not only Greg Fahy and I, but the whole cryonics community at the time was
aware of this work (everyone was actually on speaking terms and exchanging
correspondence on this very paper!). Further, an entire detailed protocol
entitled "Instructions for the Induction of Solid State Hypothermia in
Humans" was written by Fred and Linda Chamberlain which actually had
illustrations of refrigerated perfusion boxes and machines (as well as some
engineering specs and circuit diagrams) to allow for perfusion of whole
humans to -79 C. Further, Fred and Linda built a PROTOTYPE cool down
box/perfusion system for this very technique circa 1974-75!

WHY wasn't it used?  Because extensive lab work with BOTH kidney (Fahy) and
brain (me) established that what worked for taneia coli muscle did NOT wok
for these two organs/tissues. Brains in particular were creamed (literally)
by membrane toxicity, pretty much no matter what I did (massive foaming of
aerated media on DMSO removal indicating membrane lysis). Later, Greg
picked up on the use of formamide (the work of Baxter and Lathe) as what
was at first thought to be an antagonist to DMSO toxicity (actually it
turns out to be the reverse, formamide toxicity is antagonized by DMSO, a
nontrivial difference). 

We also ran into other VERY serious problems. First, as you will note,
Elford and Walter SUPERFUSED their taneia coli muscle, they did not PERFUSE
it. This is easy to do: you just pour off the liquid and add fresh CPA for
every concentration change. In fact, I remember the algorithm VERY well (I
think) having done this experiment with brain slices maybe upwards of 100
times: 20% DMSO at 0 C cool to -7 after equilibration (~1hr), substitute
30% DMSO at -7 for ~1hr, cool to -14 C and replace with 40% DMSO for ~1 hr,
cool to -39 C and substitute 50% DMSO for ~1hr, replace with 60% DMSO for
 ~1hr and cool to -79 C. 
Storage time is sharply limited by DMSO toxicity, by chilling injury
(membrane instability) and ion leakage, (even using large, impermeant, 
molecules such as PIPES). In fact, the mechanism by which PIPES works is
NOT to antagonize "cryoprotectant toxicity" at all. Rather, it prevents ion
leakage through the cell membrane which, due to hypothermia, has *both*
inactivated ion pumps and (likely) increased membrane permeability as well.
(Membrane permeability may well increase rapidly over time in the liquid
state at -79 C due to re-arrangement of the normal lamellar membrane
structure into other structures as a result of "crystallization" of
lipids.) This mechanism was not fully appreciated in 1972 when Eflord and
Walter first published. The work of Geoff Collins, David Pegg and Greg Fahy
subsequently validated the criticality of the (then so-called) "impermeant
counter-ion" inhibiting cell swelling.

The second problem is a much, much more serious one. It is easy enough to
pour out a small container of DMSO at -30 C and refill it: IF you are
patient. It is *impossible* to perfuse such a solution because of its very
high thickness at these low temperatures. For instance, a 60% DMSO solution
at -39 C is about as viscous as a good grade of pancacke syrup at room
temperature. Distribution of ultra-viscous perfusates at low temperatures
becomes virtually impossible. Art Quaife did a lovely mathematical analysis
of this problem for MANRISE TECHNICAL REVIEW many years ago. Even
relatively non-viscous perfusates such as VS4-1A used by Fahy, et al., to
attempt to vitrify kidneys can be perfused only with vast reductions of
flow at a concentration of only 50% and this at only  -20 C. 

Below that temperature, perfusion becomes impractical. Thus, *viscosity* is
a critical element in perfusate design where toxic agents are used as
cryoprotectants (CPAs). The more toxic the CPA, the lower the temperature
vs. concentration relationship has to be. This is especially true for CPAs
such as DMSO, glycol ethers and other agents capable of dissolving cell
membranes. One of the reasons glycerol has remained so attractive to
cryonicists over the years is its low ether partition co-efficient which in
theory should mean that even exposure to very high concentrations at
relatively high temperatures will not dissolve cell membranes. This is
especially important in *freezing* tissue (or people)!, see below:

Quite apart from vitrification, when a human patient, organ, tissue or cell
is subjected to freezing in the presence of CPA, the concentration of CPA
rises VERY RAPIDLY once freezing begins and typically reaches
concentrations in the 50 to 65% range at very high subzero temperatures.
So, for example, if a human cryopatient were to freeze at, say -10 C and
s/he cools at a rate of 4 C per hour, then that patient would be exposed to
MANY hours of very high CPA concentrations at temperatures intermediate
between -10 C and the solidification point, or zone of  "inhibited
toxicity" (typically below -100 C). Thus, someone perfused with say 20%
DMSO, (which will freeze at ~ -8 to -9 C)  will be exposed to
concentrations of ~40% by the time the temperature reaches ~-20 C for
*hours*. In fact, roughly 5 hours after you reach  a concentration of 40%
(assuming a cooling rate of 4 C per hour) you will be at 50% DMSO at only
-40 C !!!! 
This is plenty enough time and concentration at these temperatures to
dissolve cell membranes and other cell structures. This is one reason why
cryobiologists have been so (quite rightly) critical of cryonics since they
are exquisitely sensitive to the importance of COOLING RATE in obtaining
cell survival. This is especially an issue for lipid (and thus membrane)
solvating cryoprotetant agents such as DMSO, amides, some glycols, glycol
ethers, and all agents above a certain threshold of lipid solvating
capability.   

A brief tutorial as an aside, about 1/3rd of the average human's calorie
intake is used to maintain cell volume and control the concentration of
regulatory ions (like calcium, potassium and magnesium) in the
intracellular and extracellular spaces. Due to the high protein
concentration inside cells the intracellular environment has a net negative
charge. Present outside cells are large concentrations of positively
charged ions such as sodium and calcium. In the absence of active pumping
by the ion pumps in the cell membrane, sodium  in particular, leaks into
the cell and "carries" water with it. This results in cell swelling.
Calcium also leaks into the cell and, when the cell is rewarmed and
metabolism re-started, has the potential to wreak havoc by activating
membrane-destroying enzymes and triggering the generation of enormous free
radical activity. Long, long ago people in the organ preservation community
and the cryonics community (apparently with the exception of CI) recognized
this fact and began using buffers and carefully selected sugars and other
molecules to act as an osmotic "counterforce" or "antagonist" to the
intracellular protein: preventing or vastly slowing cell swelling until
solidification could occur. Concurrently, the concentration present in the
perfusate of calcium is decreased dramatically, and that of potassium
(which under normal conditions is actively pumped into the cell in exchange
for sodium) is increased.

The effects of the carrier solution on cryoprotectant toxicity have
subsequently been shown to be of critical importance first, I believe, by
David Pegg and his colleagues, and this continues to be a fertile area of
investigation. However, this 1972 "breakthrough" is one most knowledgeable
people in the cryonics community, at least at one time, knew and
understood. 

Mostly because its pKa is not ideal, the Zwitterionic buffer PIPES was
replaced with HEPES in the  patented perfusate used by Alcor and (at least
in the past)  ACS (under license from BioPreservation) which is designated
"MHP."  MHP has stood the test of time, and continues to perform
outstandingly well. It was also the perfusate that allowed recovery and
long-term survival, without brain damage, of dogs from 5 hours of
asanguineous perfusion and 3 hours of circulatory arrest by the progenitor
of 21st Century Medicine. As far as we know, this is the  world's record
for asanguineous perfusion. Alcor and Critical Care Resrearch, Inc. share
rights to this patent.

Since I was one of the inventors of MHP I am proud of its durability, and
note that other solutions used in successful extended dog asanguineous
perfusion by others contain the core ingredients of MHP in about the same
concentrations. I will be happier still if current research makes MHP
obsolete and vastly extends the length of time that both NORMOTHERMIC and
hypothermic preservation can be achieved in dogs an presumably other
mammals such as humans.

Yes, I said NORMOTHERMIC preservation. Doug Skrecky has challenged Thomas
Donaldson on the feasibility of near-term normothermic "suspended
animation." Doug, ever the student of the obscure, should give some thought
to the capabilities of complex organisms such as species of African eel
(among many others, including some vertebrates) which wall themselves off
in watertight "capsules" of secretions in the dried mud and tolerate 3-5
months of temperatures in excess of 43 C without the loss of significant
body mass or water, and who show very little measurable metabolic activity
(within the limits of accuracy of measurement when the work was done on
these animals in the 1960's). 

At CCRI, we currently think that 1 hour of normothermic cardiac arrest with
pre-medication is within reach, thus allowing trauma patients who are
bleeding to death time to reach tertiary care facilities where they can be
definitively treated and "re-booted."

Mike Darwin, Director of Research
Critical Care Research, Inc.

Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=12595