From: kqb@cbnewsl.cb.att.com (kevin.q.brown)
Subject: Cryoprotectant Issues
Organization: AT&T Bell Laboratories
Date: Fri, 18 Dec 1992 16:37:21 GMT
X-Crossposted-To: cryonics mailing list

    ------------------------ Forwarded Message ------------------------

> Date: 18 Dec 92 02:12:49 EST
> From: Mike Darwin <70023.3041@CompuServe.COM>
> Message-Subject: Reply to Ben Best's Reply
> Date: 13 December, 1992

     I agree that 4M glycerol should the be the minimum concentration used 
in further studies.  In fact, per Greg's and my recommendation  suspension 
patients are now being perfused with 6M glycerol as you know.

     Regarding the use of DMSO.  In principal I have no objection to  what 
you  are saying.  However, this kind of approach MUST be tried and  proven 
superior  in a relevant animal model before changes are made in the  human 
protocol.  Idle theorizing, no matter how compelling and informed, is  not 
sufficient to alter existing clinical techniques. 

     Both Greg and I have seen (and in Greg's case) have copies of  Suda's 
raw  data  for these experiments.  Suda is, to the best of  my  knowledge, 
still  alive.  Don't be too hard on Suda's work.  It was  remarkably  well 
done  and yielded more positive results than most  organ  cryopreservation 
work  from the period.  Also, many cryobiologists were slow to  appreciate 
the  need for gradual removal of CPA and the use of an osmotic  antagonist 
such as mannitol to minimize cell swelling upon deglycerolization.  

     Also,  Greg  has  to carry out the final leg  of  his  cryoprotective 
loading  procedure at subzero temperatures.  I believe he perfuses in  the 
last of the VS4 at -2xC.  Otherwise the toxicity is too great.

     In  short,  I agree with your comments completely. The point  is,  we 
need  to do the laboratory investigation  to determine the best  approach.  
This  will  only  get done with a serious commitment of  time  and  money.  
Those  who  have seen the Biopreservation/Cryovita lab will know  that  we 
have  the facilities and some of the personnel required to do  this  work.  
It  remains to be seen whether the cryonics community will be  forthcoming 
with the money.

     It  has been known for some time now that the blood brain barrier  can 
be  opened  by  "pulsing"  the  cerebral  circulation  with  a  bolus   of 
hyperosmolar solution.  I believe that the "pulse" has to be in the  range 
of  1200 mOsm, but I may be wrong about the exact number.  This is a  well 
established  technique  and is being used routinely to open  the  BBB  for 
research  purposes.  It has been applied clinically in some situations  to 
open  the BBB for delivery of chemotherapeutic agents which  normally  are 
excluded from the brain by the BBB.  I don't have a reference at hand, but 
it  shouldn't  be  hard  for  you to find one.   I  seem  to  recall  that 
SCIENTIFIC  AMERICAN  ran an article some years ago on  the  research  and 
clinical applications of this technique.

     Finally,  yes glycerol IS dehydrating the brain and other tissues  as 
well.   I would estimate that on the typical "good" case we see a  30%  to 
50%  reduction  in brain volume by the end of perfusion.  A  little  known 
fact is that the patients often look like mummies by the time perfusion is 
finished.   I have seen arms that were almost completely dehydrated --  so 
much  so  that  they literally (and this is  no  exaggeration)  looked  as 
dehydrated  as  mummies you see in a museum.  As you  might  imagine,  the 
cosmetic  effects  are not exactly good.  This is one reason  why  you  no 
longer see pictures of patients after perfusion.

     In  severe ischemic patients this effect is much less pronounced  and 
in some is absent altogether with massive edema being the case.

     As  to the signifigance of this dehydration?  Well, I'd  rather  have 
the  water  translocated  by  glycerol-induced  dehydration  than  by  ice 
formation.   At  least  the water is removed from the tissues  and  is  no 
longer present to turn into mechanically injuring ice!  Keep in mind  that 
during  freezing the cells will be dehydrated by extracellular ice in  any 
event!

     All  of this is not to say that I don't agree with you.  We  can  and 
should  find  better  CPAs.  I have identified two  that  penetrate  brain 
tissue readily and depress the freezing point and inhibit ice formation at 
least as well as DMSO.  Only time and much work will tell if these  agents 
will prove amenable to clinical application.

     As  to  the  immediate use of DMSO, keep in  mind  that  ALL  cryonic 
suspension  patients  suffer  ischemic injury prior  to  suspension.   The 
prolonged period of shock most patients experience during the agonal phase 
is  no  doubt causing severe injury to the vascular  endothelium.   Always 
remember  we  are working with medicine's leavings  and  failures.   While 
anathema  to  cryonicists it IS occasionally important  to  remember  that 
these are people who have DIED (at least by current criteria).  That is no 
small thing.  More people need to realize that.
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