X-Message-Number: 1389
Date: 03 Dec 92 06:49:03 EST
From: Paul Wakfer <>
Subject: CRYONICS: Freezing Damage (Darwin) Part 1

Note: This posting is from Mike Darwin

     There  have been several requests for information about the  kind 
of  damage done during cryonic suspension.  In particular, there  have 
been  requests for detailed, objective studies.  As a result  of  this 
interest  I have decided to post a research paper which is now in  the 
(hopefully) final stages of preparation for pulication.

     However,  there is a serious shortcoming to this posting,  namely 
that  of  necessity  there can be no accompanying  light  or  electron 
micrographs.   In this case this is a serious handicap,  although  for 
many  readers the micrographs would mean little.  In any event, it  is 
to  be  hoped  that  this  paper  can  be  prepared  for  more  formal 
publication within 6 to 12 months.  Anyone who wishes to assist me  in 
this  capacity  should  feel  free  to  do  so  (the  EM's  and  light 
micrographs need to be captioned and laid out -- a formidable task).

     Many caveats about the validity of this work are contained in its 
closing paragraphs.  However, I would like to add the following:   The 
presence  of  pericapillary  ice holes has  been  verified  by  freeze 
substitution  work done by Dr. Gregory M. Fahy of the Red Cross  Organ 
preservation  lab.  Similarly Dr. Fahy's freeze-substitution work  has 
documented  the presence of massive ice crystals which comprise  about 
60% of the tissue volume.  I gather that Dr. Fahy feels somewhat  more 
optimistic about preservation of neuronal connectivity than I do,  but 
one thing we are both agreed on: our work clearly demonstrates serious 
histogical  disruption  with tears or fractures in  the  brain  tissue 
appearing at approximately 3 to 5 micron intervals.

     I  think  it  is  also  fair  to  say  that  anyone,  layman   or 
neurophysiologist  who  looks at either the pictures in the  study  by 
Darwin,  et  al.,  or  the  pictures  generated  by  Fahy  of  freeze-
substituted  brains (showing massive histological disruption  by  ice) 
will be given pause for thought about the workability of cryonics.

     I have great admiration for Dr. Merkle and his work.  But I would 
also  point out that in the theoretical domain where there is  a  will 
there  is  almost  always a way.  Alas, the  real  world  is  somewhat 
harsher.   We  all  want and need to  believe  very  desperately  that 
cryoinjury  can  and will be reversed.  However, there  is  no  direct 
evidence  of this. The kind of damage my colleagues and I observed  in 
the  study  below is carefully "qualified," to make it  good  science.  
However,  in  this  preamble I am a bit freer and I  can  say  that  I 
believe  that the damage is at least as bad as we saw -- it  would  be 
hard  to  imagine  it being any worse short of  calling  it  a  tissue 
homogenate  --  something  you  get when you run  a  brain  through  a 
blender.   It is my gut feel that post-thaw artifactual  stirring  was 
not the main reason things look bad.  I think things look bad  because 
they *are* bad.  

     Does  that mean patients frozen with today's techniques will  not 
be  revived?  I do not know.  Does that mean we should *not*  continue 
to  freeze people?  No.  What it does mean is that we need to do  some 
serious work to improve the situation.  The kind of damage we observed 
and are observing is a consequence of ice formation.  At a minimum  we 
can  do a great deal to reduce or eliminate ice formation.   A  major, 
comprehensive research proposal is under development at this time  and 
should  be  ready  for submission to the cryonics  community  by  late 
Spring or early Summer.

     In  the  meantime  my colleagues and  I  at  Biopreservation  and 
Cryovita are working hard to make further improvements on  hypothermic 
brain  presevation  which  will  allow us  to  conduct  the  necessary 
cryopreservation work with greater ease.

     Also in order is a word about Jerry Leaf, who entered  suspension 
over  a year ago.  This work was completed in the mid 1980's and  this 
paper  was  completed  in  draft form  circa  1988.   Jerry  read  and 
commented  on the draft shortly after it was written.   Most, but  not 
all  of  his  suggested  changes were  incorporated.   The  final  two 
paragraphs  of  summary  were written after his  suspension.   I  feel 
comfortable that Jerry would want this paper published, warts and all. 
The  work that underpins it took a great deal of his time and  effort.  
Indeed,  while none of us knew it at the time, this work  comprised  a 
major  block of Jerry's cryonics-science productive life.  In  drawing 
the few conclusion I draw, I have striven to be as objective as  Jerry 
would  have  been.  I have also submitted this work for  review  by  a 
prominent   cryonicist-cryobiologist  whom  I  know  Jerry   respected 
greatly.   I have incorporated all of this cryobiologists  substantive 

     Finally,  to Jerry: may you someday have the pleasure of  reading 
these  words  and  proving  us both  wrong  about  the  prospects  for 
recovery.  Jerry, I miss you more than words can tell.  

by Michael Darwin, Jerry Leaf, Hugh L. Hixon

I.   Introduction                                   
II.  Materials and Methods
III. Effects of Glycerolization
IV.  Gross Effects of Cooling to and Rewarming From -196*C
V.   Effects of Cryopreservation on Histology of Selected Tissues
VI.  Effects of Cryopreservation on Ultrastructure of Selected Tissues
VII. Summary and Discussion


     The  immediate  goal  of cryonic suspension  is  to  use  current 
cryobiological  techniques  to  preserve the  brain  structures  which 
encode personal identity adequately enough to allow for  resuscitation 
or reconstruction of the individual should molecular nanotechnology be 
realized (1,2).  Aside from two previous isolated efforts (3,4)  there 
has  been  virtually no systematic effort to examine the  fidelity  of 
histological,  ultrastructural, or even gross structural  preservation 
of  the brain following cryopreservation in either an animal or  human 
model.    While  there  is  a  substantial  amount  of  indirect   and 
fragmentary  evidence  in the  cryobiological  literature  documenting 
varying  degrees  of  structural  preservation  in  a  wide  range  of 
mammalian tissues (5,6,7), there is little data of direct relevance to 
cryonics.   In particular, the focus of contemporary  cryobiology  has 
been   on   developing  cryopreservation  techniques   for   currently 
transplantable  organs,  and this has necessarily  excluded  extensive 
cryobiological  investigation  of  the brain, the  organ  of  critical 
importance to human identity and mentation.

     The  principal  objective of this pilot study was to  survey  the 
effects  of glycerolization, freezing to liquid nitrogen  temperature, 
and  rewarming  on  the physiology, gross  structure,  histology,  and 
ultrastructure of both the ischemic and non-ischemic adult cats  using 
a preparation protocol similar to the one then in use on human cryonic 
suspension patients.  The non-ischemic group was given the designation 
Feline Glycerol Perfusion (FGP) and the ischemic group was referred to 
as Feline Ischemic Glycerol Perfusion (FIGP). 

     The work described in this paper was carried out over a  19-month 
period from January, 1982 through July, 1983.  The perfusate  employed 
in this study was one which was being used in human cryonic suspension 
operations at that time, the composition of which is given in Table I.  
The principal cryoprotectant was glycerol.


Preperfusion Procedures

     Nine adult cats weighing between 3.4 and 6.0 kg were used in this 
study.  The animals were divided evenly into a non-ischemic and a  24-
hour mixed warm/cold ischemic group.  All animals received humane care 
in  compliance  with  the  "Principles  of  Laboratory  Animal   Care" 
formulated by the National Society for Medical Research and the "Guide 
for  the Care and Use of Laboratory Animals" prepared by the  National 
Institutes  of  Health  (NIH Publication  No.  80-23,  revised  1978).  
Anesthesia   in  both  groups  was  secured  by  the   intraperitoneal 
administration of 40 mg/kg of sodium pentobarbital.  The animals  were 
then  intubated and placed on a pressure-cycled respirator.   The  EKG 
was monitored throughout the procedure until cardiac arrest  occurred.  
Rectal and esophageal temperatures were continuously monitored  during 
perfusion using YSI type 401 thermistor probes.    

     Following placement of temperature probes, an IV was  established 
in  the medial foreleg vein and a drip of Lactated Ringer's was  begun 
to  maintain  the  patency of the IV and  support  circulating  volume 
during  surgergy. Premedication (prior to perfusion) consisted of  the 
IV  administration of 1 mg/kg of metubine iodide to inhibit  shivering 
during  external  and  extracorporeal cooling  and  420  IU/kg  sodium 
heparin  as  an anticoagulent.  Two 0.77 mm I.D.  Argyle  Medicut  15" 
Sentinel line catheters with Pharmaseal K-69 stopcocks attached to the 
luer fittings of the catheters were placed in the right femoral artery 
and vein.  The catheters were connected to Gould Model P23Db  pressure 
transducers   and  arterial  and  venous  pressures   were   monitored 
throughout the course of perfusion.

Surgical Protocol

     Following placement of the monitoring catheters, the animals were 
transferred  to a tub of crushed ice and positioned for surgery.   The 
chest  was shaved and a median sternotomy was performed.   The  aortic 
root was cleared of fat and a purse-string suture was placed,  through 
which  a  14-gauge  angiocath was introduced.   The  angiocath,  which 
served  as  the  arterial  perfusion cannula,  was  snared  in  place, 
connected  to  the  extracorporeal circuit and cleared  of  air.   The 
pericardium  was  opened  and tented to expose the  right  atrium.   A 
purse-string  suture was placed in the apex of the right atrium and  a 
USCI  type  1967 16 fr. venous cannula was introduced  and  snared  in 
place.  Backties were used on both the arterial and venous cannulae to 
secure  them and prevent accidental dislodgment during the  course  of 
perfusion.  Placement of cannulae is shown in Figure 2.

Extracorporeal Circuit

     The extracorporeal circuit (Figure 3) was of composed of 1/4" and 
3/8"  medical grade polyvinyl chloride tubing.  The circuit  consisted 
of  two  sections:  a  recirculating loop  to  which  the  animal  was 
connected  and a glycerol addition system.  The  recirculating  system 
consisted  of  a  10 liter polyethylene reservoir  positioned  atop  a 
magnetic  stirrer, an arterial (recirculating) roller pump,  an  Erika 
HPF-200  hemodialyzer which was used as a hollow fiber oxygenator  (8) 
(or alternatively, a Sci-Med Kolobow membrane oxygenator), a  Travenol 
Miniprime  pediatric  heat  exchanger, and a 40-micron  Pall  LP  1440 
pediatric blood filter.  The recirculating reservoir was  continuously 
stirred with a 2" teflon-coated magnetic stir bar driven by a  Corning 
PC  353 magnetic stirrer.  Temperature was continuously  monitored  in 
the  arterial line approximately six inches from the arterial  cannula 
using a Sarns in-line thermistor temperature probe and YSI 42SL remote 
sensing  thermometer.  Glycerol concentrate was continuously added  to 
the the recirculating system using a Drake-Willock hemodialysis pump.  

Storage and Reuse of the Extracorporeal Circuit

     After  use the circuit was flushed extensively with filtered  tap 
and distilled water, and then flushed and filled with 3%  formaldehyde 
in distilled water to prevent bacterial overgrowth.  Prior to use  the 
circuit was again thoroughly flushed with filtered tap water, and then 
with  filtered distilled water (including both blood and gas sides  of 
the hollow fiber dialyzer; Kolobow oxygenators were not re-used).   At 
the  end  of  the distilled water flush, a test for  the  presence  of 
residual formaldehyde was performed using Schiff's Reagent.  Prior  to 
loading  of  the perfusate, the circuit was rinsed with 10  liters  of 
clinical  grade normal saline to remove any particulates  and  prevent 
osmotic dilution of the base perfusate.

     Pall filters and arterial cannula were not re-used.  The  circuit 
was replaced after a maximum of three uses.

Preparation of Control Animals

Fixative Perfused

     Two control animals were prepared as per the above.  However, the 
animals  were subjected to fixation after induction of anesthesia  and 
placement  of cannulae.  Fixation was achieved by first perfusing  the 
animals   with  500  cc  of  bicarbonate-buffered  Lactated   Ringer's 
containing 50 g/l hydroxyethyl starch (HES) with an average  molecular 
weight  of  400,000 to 500,000 supplied by  McGaw  Pharmaceuticals  of 
Irvine, Ca (pH adjusted to 7.4) to displace blood and facilitate  good 
distribution of fixative, followed immediately by perfusion of 1 liter 
of  modified  Karnovsky's  fixative (Composition given  in  Table  I).  
Buffered Ringers-HES perfusate and Karnovsky's solution were  filtered 
through 0.2 micron filters and delivered with the same  extracorporeal 
circuit described above.  

     Immediately   following  fixative  perfusion  the  animals   were 
dissected and 4-5 mm thick coronal sections of organs were cut, placed 
in  glass screw-cap bottles, and transported, as detailed  below,  for 
light or electron microscopy.

Straight Frozen Non-ischemic Control

     One animal was subjected to straight freezing (i.e., not  treated 
with   cryoprotectant).    Following  induction  of   anesthesia   and 
intubation  the  animal  was supported on  a  respirator  while  being 
externally  cooled  in  a  crushed  ice-water  bath.   When  the   EKG 
documented  profound bradycardia at 26*C, the animal was  disconnected 
from  the  respirator,  placed  in a  plastic  bag,  submerged  in  an 
isopropanol  cooling bath at -10*C, and chilled to dry ice and  liquid 
nitrogen  temperature  per the same protocol used for  the  other  two 
experimental groups as described below.

Preparation of FGP Animals

     Following  placement of cannulae, FGP animals were  subjected  to 
total  body  washout  (TBW) by open-circuit perfusion  of  500  cc  of 
glycerol-free  perfusate.  The extracorporeal circuit was then  closed 
and constant-rate addition of glycerol-containing perfusate was begun.  
Cryoprotective  perfusion continued until the target concentration  of 
glycerol  was reached or the supply of glycerol-concentrate  perfusate 
was exhausted.

Preparation of FIGP Animals

     In   the  FIGP  animals,  respirator  support  was   discontinued 
following anasthesia and administration of Metubine.  The endotracheal 
tube was clamped and the ischemic episode was considered to have begun 
when cardiac arrest was documented by absent EKG.

     After the start of the ischemic episode the animals were  allowed 
to  remain on the operating table at room temperature ( 22*C to  25*C) 
for  a  30  minute period to simulate  the  typical  interval  between 
pronouncement  of legal death in a clinical environment and the  start 
of  external cooling at that time.  During the 30 minute  normothermic 
ischemic  interval the femoral cut-down was performed  and  monitoring 
lines were placed in the right femoral artery and vein as per the  FGP 
animals.  Prior to placement, the monitoring catheters were  irrigated 
with normal saline, and following placement the catheters were  filled 
with 1000 unit/cc of sodium heparin to guard against clot  obstruction 
of the catheter during the post-mortem ischemic period.

     After the 30 minute normothermic ischemic period the animals were 
placed  in  a  1-mil polyethylene bag,  transferred  to  an  insulated 
container  in  which  a bed of crushed ice had  been  laid  down,  and 
covered  over with ice.  A typical cooling curve for a FIGP animal  is 
presented in Figure 1. FIGP animals were stored on ice in this fashion 
for a period of 24 hours, after which time they were removed from  the 
container and prepared for perfusion using the surgical and  perfusion 
protocol described above.


     The  perfusate  was an intracellular formulation  which  employed 
sodium  glycerophosphate  as the impermeant species  and  hydroxyethyl 
starch  (HES)(av.  MW   400,000  -  500,000)  as  the  colloid.    The 
composition of the base perfusate is given in Table I.  The pH of  the 
perfusate  was adjusted to 7.6 with potassium hydroxide.  A  pH  above 
7.7, which would have been "appropriate" to the degree of  hypothermia 
experienced  during cryoprotective perfusion (9), was  not  achievable 
with  this mixture owing to problems with complexing of magnesium  and 
calcium   with  the  phosphate  buffer,  resulting  in  an   insoluble 

     Perfusate components were reagent or USP grade and were dissolved 
in USP grade water for injection.  Perfusate was prefiltered through a 
Whatman GFB glass filter (a necessary step to remove precipitate)  and 
then passed through a Pall 0.2 micron filter prior to loading into the 
extracorporeal circuit.

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