X-Message-Number: 4137
Date: 03 Apr 95 03:15:03 EDT
From: Mike Darwin <>
Subject: SCI.CRYONICS Freezing and memory

Bruce Zimov makes some very good comments about cerebral ischemia and refers to
a book by Lutz, et al.  Most of his statements are correct, however
excitotoxicity due to NMDA receptor activation is not, in our hands or in the
hands of most others, a signifigant cause of ischemic or ischemia-reperfusion
injury in models of global brain ischemia.  We have tried NMDA receptor

inhibitors in a canine cerebeal ischemia (cardiac arrest) model with not only no
success, but with worse results than no drugs.

Excitoxcicity does occur however, and we are currently looking hard at kainate
and quisqualate receptor blockers as well as others which, for priprietary
reasons, I cannot talk about publically.  I can tell you that we have recovered
one dog named Jake after 10 minutes of cardiac arrest at 37.5xC followed by 5
minutes of reperfusion (using bypass) at a mean arterial pressure (MAP) of 155
mmHg or less and ANOTHER 5 minutes of reperfusion at a MAP of 30 mmHg or less,
followed by hypertensive , (MAP =140 mmHg) hypothermic reperfusion.  Jake was
blind for about 2 weeks and is still a little clumsy 3 months later.  But, he

know his name( (and he knew it during ICU care), he can still learn (albeit more
slowly). His only visible defect is that he will run in circles to the left if
you put him in a small pen or stress him with confinement: otherwise he is

normal with a good overall performce catergory (OPC) score (we use Peter Safar's
system and Jake scores a 5).


Reperfusion injury has concerned me for a long time, and we administer over half
a dozen drugs to our cryopreservation patients after beginning CPR following

cardiac arrest.  We also put people on PREmedication to protect against cerebral
ischemia when they become terminally ill.  For instance, some drugs such as
Dilantin (phenytoin), are very cerebroprotective if given *before* before the
insult, but don't show much utility if given afterwards.  This a complex area
and really beyond the scope of discussion here.

However, I wish to make one more point: while cerebral ischemia and reperfusion
injury concern me greatly and mitigating them has occupied much of my career in
cryonics, I am far more concerned about cryoinjury.  Indeed, I now feel that in
patients who retain good cerebral perfusion up until the time of cardiac arrest
the ischemic intervals they are experiencing in an optimum setting (3-10

minutes) are not irreversible given the technology that we (Biopreservation) has
right now and is developing (Jake is living proof of this).  

However, once you've seen some EMs from frozen-thawed brains, even brains
treated with high concentration of cryoprotectant, you will realize that insult
from ischemia is the LEAST of the patients' problems.  I've described
frozen-thawed cryoprotected human brains as chopped steak.  I wasn't kidding.
The injury is tremenous and one of its most disturbing features is the
reorganization of intracellular structures in membranous vesicles.  Indeed,

finding intact brain cell membranes becomes a challenge.  Long processes are cut
and chopped by ice and many axons are empty or are just feilds of debris.  Of
course, this is post-thaw, post fixation, post a lot of things... and much of
the stirring of the pieces may have occured on thawing.  However, we now have
freeze-substitution pictures of brains in the frozen state after 4M glycerol
perfusion (we're getting ready to do similar studies with 7.4M and 1.5M) and it
isn't a pretty picture.

My point here is that at an EM level things are a mess.  To put things in
perspective I'll use the following analogy:

Take a picture of Kobe Japan from space (say 200 miles up) BEFORE the
earthquake.  Take on afterwards (and after the *fires are out*).  It will be
very hard to see the changes, but you can, if you look closely notice that the
gridwork and order of things is a little fuzzy and what were once clean lines
(like the bullet train track) have litle breaks in them.  But on the whole
things look very good.  This is *exactly* what you see when you look at brains
at the light level.

Now overfly Kobe at say 10,000 feet. Damage is visible everywehere.  Building
are collapsed, communications lines cut, water is flowing from broken pipes and

roadways are disrupted.  None of this is visible at 200 miles up.  Finally, walk
around on the ground with a camera.  Now you can really see the havoc!

How does this relate to brain cryoinjury.  Well, my suspicion is that gross

personality traits, temperament and some skills (such as walking) are encoded on

a level comparable to that seen with light microscopy.  More specific procedural
memories are probably encoded at the level of complexity comparable to an
overflight at 10K feet.  And declarative memories are encoded at the ground
level (synapse/receptor/membrane morphology changes).  I think contemporary
cryopreservation patients' general coarse wiring diagram may be inferrable, but
not easily, since there are tears and major disruption of connection at 10-30
micron intervals (these are visible at even the light level) throughout all

three dimensions of the brain.  When we get down to the ultrastructural level of
seeing membranes as lines on an EM things are really messed up. As to the
integrity of boutons and vesicles involved in hypothetical models of learning
mediated changes of synapses: well we haven't looked, but I'd be very surprized
if the news was good; especially when I see amorphous granular debris scattered

in ice holes, major morpholohical changes in intracellular and plasma membranes,

and so on.  Synapses: yeah, you still see 'em, they're pretty rugged.  But thats

often all you see is a synapse just sitting out there in field of debris.  Empty
remanents of axons with unravelled, swollen mylein, ice holes, naked nucei with
fragments of endothial cells still hanging on them in capillaries are all par
for the course.


When you look at ice in a frozen brain, you come to understand WHY these changes
have occured.  Maybe we will be able to infer things in frozen patient by
dismantling them in the frozen state.  But this is not all clear to me and many
of the membrane changes may be going on during freezing when the membrane
architecture is being reshaped by cooling and by exposure to an extremely
hyperosmolar environment.  Inferring the normal state from those changes is a
situation I doubt is possible given our current understanding of physical law.

This why I am so focused on improving brain cryopreservation and in looking at

brains frozen with current techniques after thawing *and in the frozen state* in
order to tell exactly what's happening.  The only major modality we have *not*

been able to bring to bear is the use of freeze-fracture which we cannot seem to
find any lab competent to do.  This is very important because it would allow us
to look at membrane morphology closer to the "street level"  (and in three

dimension) than we have ever been able to do so far.  Anyone who can point me to

a good EM center that does free lance freeze-fracture work of high quality would
be greatly appreciated.

Mike Darwin

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