X-Message-Number: 5460
Date: 21 Dec 95 17:32:20 EST
From: Mike Darwin <>
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


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